Area de Genomica y Salud

Valencia, Spain

Area de Genomica y Salud

Valencia, Spain
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Garcia-Lopez R.,Area de Genomica y Salud | Garcia-Lopez R.,University of Valencia | Garcia-Lopez R.,CIBER ISCIII | Moya A.,Area de Genomica y Salud | And 6 more authors.
Clinical Otolaryngology | Year: 2014

Objective: This study aimed to survey the presence of known oncoviruses in oral biopsies from patients diagnosed with the aetiologically undetermined proliferative verrucous leukoplakia and compare results to those from milder oral leukoplakia (OL) cases, oral squamous cell carcinoma, a common outcome of the lesions of interest, and healthy controls. Design: Blind, retrospective, case-control study. Setting: A stomatology unit in an academic Hospital and a Public Health laboratory. Participants: Forty patients were divided in four groups. Ten patients had been diagnosed with proliferative verrucous leukoplakia, 10 with OL and 10 with OSCC, and 10 were healthy subjects. Main outcome measures: The presence or absence of oncovirus DNA was assayed with the amplification of viral genetic markers using PCR and subsequent gel electrophoresis confirmation. Amplified fragments were sequenced and identified bioinformatically. Results: No DNA from the herpesvirus, papillomavirus or polyomavirus species was detected in the samples. Conclusions: No association between proliferative verrucous leukoplakia and target viruses was detected. A higher throughput viral metagenomic approach may prove valuable for future analyses, as it would not be restricted to a priori knowledge of potential targets. © 2014 John Wiley & Sons Ltd.

Dzunkova M.,Area de Genomica Y Salud | Dzunkova M.,University of Valencia | Dzunkova M.,CIBER ISCIII | Garcia-Garcera M.,Area de Genomica Y Salud | And 11 more authors.
PLoS ONE | Year: 2014

The large amount of DNA needed to prepare a library in next generation sequencing protocols hinders direct sequencing of small DNA samples. This limitation is usually overcome by the enrichment of such samples with whole genome amplification (WGA), mostly by multiple displacement amplification (MDA) based on φ29 polymerase. However, this technique can be biased by the GC content of the sample and is prone to the development of chimeras as well as contamination during enrichment, which contributes to undesired noise during sequence data analysis, and also hampers the proper functional and/or taxonomic assignments. An alternative to MDA is direct DNA sequencing (DS), which represents the theoretical gold standard in genome sequencing. In this work, we explore the possibility of sequencing the genome of Escherichia coli fs 24 from the minimum number of DNA molecules required for pyrosequencing, according to the notion of one-bead-one-molecule. Using an optimized protocol for DS, we constructed a shotgun library containing the minimum number of DNA molecules needed to fill a selected region of a picotiterplate. We gathered most of the reference genome extension with uniform coverage. We compared the DS method with MDA applied to the same amount of starting DNA. As expected, MDA yielded a sparse and biased read distribution, with a very high amount of unassigned and unspecific DNA amplifications. The optimized DS protocol allows unbiased sequencing to be performed from samples with a very small amount of DNA. © 2014 Džunková et al.

Dzunkova M.,Area de Genomica y Salud | Dzunkova M.,University of Valencia | Dzunkova M.,CIBER ISCIII | D'Auria G.,Area de Genomica y Salud | And 5 more authors.
Frontiers in Microbiology | Year: 2015

The sequence assembly of the human gut virome encounters several difficulties. A high proportion of human and bacterial matches is detected in purified viral samples. Viral DNA extraction results in a low DNA concentration, which does not reach the minimal limit required for sequencing library preparation. Therefore, the viromes are usually enriched by whole genome amplification (WGA), which is, however, prone to the development of chimeras and amplification bias. In addition, as there is a very wide diversity of gut viral species, very extensive sequencing efforts must be made for the assembling of whole viral genomes. We present an approach to improve human gut virome assembly by employing a more precise preparation of a viral sample before sequencing. Particles present in a virome previously filtered through 0.2 μm pores were further divided into groups in accordance with their size and DNA content by fluorescence activated cell sorting (FACS). One selected viral fraction was sequenced excluding the WGA step, so that unbiased sequences with high reliability were obtained. The DNA extracted from the 314 viral particles of the selected fraction was assembled into 34 contigs longer than 1,000 bp. This represents an increase to the number of assembled long contigs per sequenced Gb in comparison with other studies where non-fractioned viromes are sequenced. Seven of these contigs contained open reading frames (ORFs) with explicit matches to proteins related to bacteriophages. The remaining contigs also possessed uncharacterized ORFs with bacteriophage-related domains. When the particles that are present in the filtered viromes are sorted into smaller groups by FACS, large pieces of viral genomes can be recovered easily. This approach has several advantages over the conventional sequencing of non-fractioned viromes: non-viral contamination is reduced and the sequencing efforts required for viral assembly are minimized. © 2015 Džunková, D'Auria and Moya.

Perez-Brocal V.,Area de Genomica y Salud | Perez-Brocal V.,CIBER ISCIII | Perez-Brocal V.,University of Valencia | Garcia-Lopez R.,Area de Genomica y Salud | And 11 more authors.
Clinical and Translational Gastroenterology | Year: 2013

Objectives: This study aimed to analyze and compare the diversity and structure of the viral and microbial communities in fecal samples from a control group of healthy volunteers and from patients affected by Crohn's disease (CD).METHODS:Healthy adult controls (n=8) and patients affected by ileocolic CD (n=11) were examined for the viral and microbial communities in their feces and, in one additional case, in the intestinal tissue. Using two different approaches, we compared the viral and microbial communities in several ways: by group (patients vs. controls), entity (viruses vs. bacteria), read assembly (unassembled vs. assembled reads), and methodology (our approach vs. an existing pipeline). Differences in the viral and microbial composition, and abundance between the two groups were analyzed to identify taxa that are under-or over-represented.RESULTS:A lower diversity but more variability between the CD samples in both virome and microbiome was found, with a clear distinction between groups based on the microbiome. Only ≈5% of the differential viral biomarkers are more represented in the CD group (Synechococcus phage S CBS1 and Retroviridae family viruses), compared with 95% in the control group. Unrelated patterns of bacteria and bacteriophages were observed.CONCLUSIONS:Our use of an extensive database is critical to retrieve more viral hits than in previous approaches. Unrelated patterns of bacteria and bacteriophages may be due to uneven representation of certain viruses in databases, among other factors. Further characterization of Retroviridae viruses in the CD group could be of interest, given their links with immunodeficiency and the immune responses. To conclude, some methodological considerations underlying the analysis of the viral community composition and abundance are discussed. © 2013 the American College of Gastroenterology All rights reserved.

Albuquerque L.,University of Coimbra | Kowalewicz-Kulbat M.,University of Lodz | Drzewiecka D.,University of Lodz | Staczek P.,University of Lodz | And 5 more authors.
Systematic and Applied Microbiology | Year: 2016

Two halophilic archaea, designated strains WSM-64T and WSM-66, were isolated from a sample taken from a borehole in the currently unexploited Barycz mining area belonging to the "Wieliczka" Salt Mine Company, in Poland. Strains are red pigmented and form non-motile cocci that stain Gram-negative. Strains WSM-64T and WSM-66 showed optimum growth at 40 °C, in 20% NaCl and at pH 6.5-7.5. The strains were facultative anaerobes. The major polar lipids of the two strains were phosphatidylglycerol (PG2), phosphatidylglycerol phosphate methyl ester (PGP-Me) and sulfated diglycosyl diether (S-DGD). Menaquinone MK-8 was the major respiratory quinone. The DNA G+C content of strain WSM-64T was 61.2 mol% by HPLC method; 61.0 mol% by genome sequencing. Analysis of the almost complete 16S rRNA gene sequence indicated that the strains WSM-64T and WSM-66 (99.7% identity) represented a member of the genus Halorhabdus in the family Halobacteriaceae. Both strains formed a distinct cluster and were most closely related to Halorhabdus tiamatea SARL4BT and Halorhabdus utahensis AX-2T (DSM 12940T) (95.4% and 95.6%, respectively). ANI values of WSM-64T with the closest relative type strains were <78.5%. Based on 16S rRNA gene sequence and whole genome analyses, physiological and biochemical characteristics we describe a new species represented by strain WSM-64T (=DSM 29498T =CECT 8673T) for which we propose the name Halorhabdus rudnickae sp. nov. © 2015 Elsevier GmbH.

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