Publications by authors named "Oscar Fornas"

9 Publications

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Benchmarking of single-virus genomics: a new tool for uncovering the virosphere.

Environ Microbiol 2020 Dec 28. Epub 2020 Dec 28.

Department of Physiology, Genetics, and Microbiology, University of Alicante, Alicante, Spain.

Metagenomics and single-cell genomics have enabled the discovery of relevant uncultured microbes. Recently, single-virus genomics (SVG), although still in an incipient stage, has opened new avenues in viral ecology by allowing the sequencing of one single virus at a time. The investigation of methodological alternatives and optimization of existing procedures for SVG is paramount to deliver high-quality genomic data. We report a sequencing dataset of viral single-amplified genomes (vSAGs) from cultured and uncultured viruses obtained by applying different conditions in each SVG step, from viral preservation and novel whole-genome amplification (WGA) to sequencing platforms and genome assembly. Sequencing data showed that cryopreservation and mild fixation were compatible with WGA, although fresh samples delivered better genome quality data. The novel TruPrime WGA, based on primase-polymerase features, and WGA-X employing a thermostable phi29 polymerase, were proven to be with sufficient sensitivity in SVG. The Oxford Nanopore (ON) sequencing platform did not provide a significant improvement of vSAG assembly compared to Illumina alone. Finally, the SPAdes assembler performed the best. Overall, our results represent a valuable genomic dataset that will help to standardized and advance new tools in viral ecology.
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http://dx.doi.org/10.1111/1462-2920.15375DOI Listing
December 2020

Flow Sorting Enrichment and Nanopore Sequencing of Chromosome 1 From a Chinese Individual.

Front Genet 2019 9;10:1315. Epub 2020 Jan 9.

Genomics Core Facility, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Parc de Recerca Biomèdica de Barcelona (PRBB), Barcelona, Spain.

Sorting of individual chromosomes by Flow Cytometry (flow-sorting) is an enrichment method to potentially simplify genome assembly by isolating chromosomes from the context of the genome. We have recently developed a workflow to sequence native, unamplified DNA and applied it to the smallest human chromosome, the Y chromosome. Here, we modify improve upon that workflow to increase DNA recovery from chromosome sorting as well as sequencing yield. We apply it to sequence and assemble the largest human chromosome - chromosome 1 - of a Chinese individual using a single Oxford Nanopore MinION flow cell. We generate a selective and highly continuous assembly whose continuity reaches into the order of magnitude of the human reference GRCh38. We then use this assembly to call candidate structural variants against the reference and find 685 putative novel SV candidates. We propose this workflow as a potential solution to assemble structurally complex chromosomes, or the study of very large plant or animal genomes that might challenge traditional assembly strategies.
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http://dx.doi.org/10.3389/fgene.2019.01315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6962354PMC
January 2020

Ecogenomics of the SAR11 clade.

Environ Microbiol 2020 05 25;22(5):1748-1763. Epub 2019 Dec 25.

Evolutionary Genomics Group, División de Microbiología, Universidad Miguel Hernández, Apartado 18, San Juan 03550, Alicante, Spain.

Members of the SAR11 clade, despite their high abundance, are often poorly represented by metagenome-assembled genomes. This fact has hampered our knowledge about their ecology and genetic diversity. Here we examined 175 SAR11 genomes, including 47 new single-amplified genomes. The presence of the first genomes associated with subclade IV suggests that, in the same way as subclade V, they might be outside the proposed Pelagibacterales order. An expanded phylogenomic classification together with patterns of metagenomic recruitment at a global scale have allowed us to define new ecogenomic units of classification (genomospecies), appearing at different, and sometimes restricted, metagenomic data sets. We detected greater microdiversity across the water column at a single location than in samples collected from similar depth across the global ocean, suggesting little influence of biogeography. In addition, pangenome analysis revealed that the flexible genome was essential to shape genomospecies distribution. In one genomospecies preferentially found within the Mediterranean, a set of genes involved in phosphonate utilization was detected. While another, with a more cosmopolitan distribution, was unique in having an aerobic purine degradation pathway. Together, these results provide a glimpse of the enormous genomic diversity within this clade at a finer resolution than the currently defined clades.
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http://dx.doi.org/10.1111/1462-2920.14896DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7318151PMC
May 2020

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

Cell Rep 2019 07;28(2):352-367.e9

Departament de Biologia Cel.lular, Fisiologia i Immunologia, Universitat Autònoma de Barcelona (UAB), Cerdanyola del Vallès 08193, Spain; Genome Integrity and Instability Group, Institut de Biotecnologia i Biomedicina (IBB), Universitat Autònoma de Barcelona (UAB), Cerdanyola del Vallès 08193, Spain. Electronic address:

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.
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http://dx.doi.org/10.1016/j.celrep.2019.06.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6635386PMC
July 2019

Selective single molecule sequencing and assembly of a human Y chromosome of African origin.

Nat Commun 2019 01 2;10(1). Epub 2019 Jan 2.

Institut de Biologia Evolutiva, (CSIC-Universitat Pompeu Fabra), PRBB, Doctor Aiguader 88, Barcelona, Catalonia, 08003, Spain.

Mammalian Y chromosomes are often neglected from genomic analysis. Due to their inherent assembly difficulties, high repeat content, and large ampliconic regions, only a handful of species have their Y chromosome properly characterized. To date, just a single human reference quality Y chromosome, of European ancestry, is available due to a lack of accessible methodology. To facilitate the assembly of such complicated genomic territory, we developed a novel strategy to sequence native, unamplified flow sorted DNA on a MinION nanopore sequencing device. Our approach yields a highly continuous assembly of the first human Y chromosome of African origin. It constitutes a significant improvement over comparable previous methods, increasing continuity by more than 800%. Sequencing native DNA also allows to take advantage of the nanopore signal data to detect epigenetic modifications in situ. This approach is in theory generalizable to any species simplifying the assembly of extremely large and repetitive genomes.
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http://dx.doi.org/10.1038/s41467-018-07885-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6315018PMC
January 2019

Single-cell genomics uncover Pelagibacter as the putative host of the extremely abundant uncultured 37-F6 viral population in the ocean.

ISME J 2019 01 18;13(1):232-236. Epub 2018 Sep 18.

Department of Physiology, Genetics, and Microbiology, University of Alicante, Alicante, Spain.

The identification of relevant virus-host pairs that globally account for a large pool of carbon and nutrients in the ocean is paramount to build accurate ecological models. A previous work using single-virus genomics led to the discovery of the uncultured single-virus vSAG 37-F6, originally sorted from the Mediterranean Sea (Blanes Bay Microbial Observatory), that represents one of the most abundant dsDNA viral population in the marine surface virosphere. Here, from same sampling site, we report that a Pelagibacter single-cell contained a viral member of vSAG 37-F6 population, by means of PCR screening of sorted, genome-amplified single cells with vSAG 37-F6-specific primers and whole-genome sequencing. Furthermore, viruses from this population were also found in three other Pelagibacter single cells from the South Pacific and Atlantic oceans. These new uncultured pelagiphages were genetically different from the previously characterized pelagiphage isolates. Data showed that the uncultured vSAG 37-F6 population represents the Pelagibacter phages that inhabit the sunlit ocean better, and contains a vast unrecognized microdiversity.
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http://dx.doi.org/10.1038/s41396-018-0278-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6299107PMC
January 2019

Deciphering the Human Virome with Single-Virus Genomics and Metagenomics.

Viruses 2018 03 6;10(3). Epub 2018 Mar 6.

Department of Physiology, Genetics, and Microbiology, University of Alicante, 03690 Alicante, Spain.

Single-cell genomics has unveiled the metabolic potential of dominant microbes inhabiting different environments, including the human body. The lack of genomic information for predominant microbes of the human body, such as bacteriophages, hinders our ability to answer fundamental questions about our viral communities. Here, we applied single-virus genomics (SVGs) to natural human salivary samples in combination with viral metagenomics to gain some insights into the viral community structure of the oral cavity. Saliva samples were processed for viral metagenomics ( = 15) and SVGs ( = 3). A total of 1328 uncultured single viruses were sorted by fluorescence-activated virus sorting followed by whole genome amplification. Sequencing of 24 viral single amplified genomes (vSAGs) showed that half of the vSAGs contained viral hallmark genes. Among those bona fide viruses, the uncultured single virus 92-C13 putatively infecting oral Streptococcus-like species was within the top ≈10 most abundant viruses in the oral virome. Viral gene network and viral metagenomics analyses of 439 oral viruses from cultures, metagenomics, and SVGs revealed that salivary viruses were tentatively structured into ≈200 major viral clusters, corresponding to approximately genus-level groupings. Data showed that none of the publicly available viral isolates, excepting an Actinomyces phage, were significantly abundant in the oral viromes. In addition, none of the obtained viral contigs and vSAGs from this study were present in all viromes. Overall, the data demonstrates that most viral isolates are not naturally abundant in saliva, and furthermore, the predominant viruses in the oral cavity are yet uncharacterized. Results suggest a variable, complex, and interpersonal viral profile. Finally, we demonstrated the power of SVGs in combination with viral metagenomics to unveil the genetic information of the uncultured viruses of the human virome.
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http://dx.doi.org/10.3390/v10030113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5869506PMC
March 2018

Single-virus genomics reveals hidden cosmopolitan and abundant viruses.

Nat Commun 2017 06 23;8:15892. Epub 2017 Jun 23.

Department of Physiology, Genetics, and Microbiology, University of Alicante, Carretera San Vicente del Raspeig, San Vicente del Raspeig, Alicante 03690, Spain.

Microbes drive ecosystems under constraints imposed by viruses. However, a lack of virus genome information hinders our ability to answer fundamental, biological questions concerning microbial communities. Here we apply single-virus genomics (SVGs) to assess whether portions of marine viral communities are missed by current techniques. The majority of the here-identified 44 viral single-amplified genomes (vSAGs) are more abundant in global ocean virome data sets than published metagenome-assembled viral genomes or isolates. This indicates that vSAGs likely best represent the dsDNA viral populations dominating the oceans. Species-specific recruitment patterns and virome simulation data suggest that vSAGs are highly microdiverse and that microdiversity hinders the metagenomic assembly, which could explain why their genomes have not been identified before. Altogether, SVGs enable the discovery of some of the likely most abundant and ecologically relevant marine viral species, such as vSAG 37-F6, which were overlooked by other methodologies.
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http://dx.doi.org/10.1038/ncomms15892DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5490008PMC
June 2017

Flow cytometric-based isolation of nucleated erythroid cells during maturation: an approach to cell surface antigen studies.

Cytometry 2002 Dec;50(6):305-12

Department of Hematotherapy, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain.

Nucleated red blood cells (NRBCs) are involved in normal physiologic processes, as well as in several malignancies. They are usually counted manually under the microscope. However, blood sample manipulation may be a source of variability and manual counting is imprecise, time-consuming, and subjective. To improve identification of CD45-negative cells, we used a flow cytometry technique that avoids the addition of lysing reagents and stains viable cell nuclei. We applied this method for counting and isolating NRBC subpopulations in whole blood samples, using DNA/RNA viable staining to discriminate nonnucleated erythroid cells and debris. NRBC counts gave 197.95 cells per mm(3) in mobilized peripheral blood samples (1.00%, n = 20), 3897.59 cells per mm(3) in leukapheresis products (3.08%, n = 20), and 765.21 cells per mm(3) in cord blood samples (6.09%, n = 20). Normal bone marrow counts were 5449.42 cells per mm(3) (11.76%, n = 20). Scatter profiles showed three distinct populations, from early to late-stage erythroblasts, consisting of erythroblasts, orthochromatic erythroblasts, and ejected nuclei, as confirmed by Wright-Giemsa staining. In addition, flow cytometry immunophenotyping showed that glycophorin A was expressed dimly on NRBCs during maturation. These findings point to the feasibility of live NRBCs studies, which offer great potential for a wide range of disciplines.
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http://dx.doi.org/10.1002/cyto.10158DOI Listing
December 2002