Cantanhede, Portugal
Cantanhede, Portugal

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Cerqueira T.,University of The Azores | Pinho D.,Next Generation Sequencing Unit | Froufe H.,Next Generation Sequencing Unit | Santos R.S.,University of The Azores | And 3 more authors.
Microbial Ecology | Year: 2017

Menez Gwen, Lucky Strike and Rainbow are the three most visited and well-known deep-sea hydrothermal vent fields in the Azores region, located in the Mid-Atlantic Ridge. Their distinct geological and ecological features allow them to support a diversity of vent communities, which are largely dependent on Bacteria and Archaea capable of anaerobic or microaerophilic metabolism. These communities play important ecological roles through chemoautotrophy, feeding and in establishing symbiotic associations. However, the occurrence and distribution of these microbes remain poorly understood, especially in deep-sea sediments. In this study, we provide for the first time a comparative survey of the sediment-associated microbial communities from these three neighbouring vent fields. Sediment samples collected in the Menez Gwen, Lucky Strike and Rainbow vent fields showed significant differences in trace-metal concentrations and associated microbiomes. The taxonomic profiles of bacterial, archaeal and eukaryotic representatives were assessed by rRNA gene-tag pyrosequencing, identified anaerobic methanogens and microaerobic Epsilonproteobacteria, particularly at the Menez Gwen site, suggesting sediment communities potentially enriched in sub-seafloor microbes rather than from pelagic microbial taxa. Cosmopolitan OTUs were also detected mostly at Lucky Strike and Rainbow sites and affiliated with the bacterial clades JTB255, Sh765B-TzT-29, Rhodospirillaceae and OCS155 marine group and with the archaeal Marine Group I. Some variations in the community composition along the sediment depth were revealed. Elemental contents and hydrothermal influence are suggested as being reflected in the composition of the microbial assemblages in the sediments of the three vent fields. Altogether, these findings represent valuable information for the understanding of the microbial distribution and potential ecological roles in deep-sea hydrothermal fields. © 2017 Springer Science+Business Media New York


Hollatz C.,University of Minho | Leite B.R.,University of Minho | Lobo J.,New University of Lisbon | Froufe H.,Next Generation Sequencing Unit | And 2 more authors.
Genome | Year: 2017

In marine and estuarine benthic communities, the inventory and estimation of species richness are often hampered by the need for broad taxonomic expertise across several phyla. The use of DNA metabarcoding has emerged as a powerful tool for the fast assessment of species composition in a diversity of ecological communities. Here, we tested the amplification success of five primer sets targeting different COI-5P regions by 454 pyrosequencing to maximize the recovery of two simulated macrobenthic communities containing 21 species (SimCom1 and SimCom 2). Species identification was first performed against a compiled reference library of macrobenthic species. Reads with similarity results to reference sequences between 70% and 97% were then submitted to GenBank and BOLD to attempt the identification of concealed species in the bulk sample. The combination of at least three primer sets was able to recover more species than any primer set alone, achieving 85% of represented species in SimCom1 and 76% in SimCom2. Our approach was successful to detect low-frequency specimens, as well as concealed species, in the bulk sample, indicating the potential for the application of this approach on marine bioassessment and inventory, including the detection of "hidden" biodiversity that would hardly be possible based on morphology only. © 2017 Published by NRC Research Press.


Cardoso J.M.S.,University of Coimbra | Fonseca L.,University of Coimbra | Gomes P.,Next Generation Sequencing Unit | Egas C.,Next Generation Sequencing Unit | Abrantes I.,University of Coimbra
Forest Pathology | Year: 2015

In this study, a cDNA sequence corresponding to the Bursaphelenchus xylophilus unc-87 homolog gene (Bx-unc-87) was identified in B. xylophilus transcriptome and characterized. This cDNA has an 1152-bp-long open-reading frame that is putatively translated into a 383 amino acids peptide. The deduced protein (Bx-UNC-87) contains highly conserved regions of the calponin-like proteins and has high sequence identity to the calponin proteins of other nematodes. In Caenorhabditis elegans, the calponin protein encoded by gene unc-87 is required to maintain the structure of myofilaments in muscle cells of the body wall. The silencing of Bx-unc-87 gene was performed by RNAi, soaking the nematodes in a solution containing dsRNA of this gene. In addition to a no dsRNA control, a non-homologous dsRNA control was also included. The Bx-unc-87 transcription was downregulated in nematodes treated with Bx-unc-87 dsRNA in comparison with the non-dsRNA-treated nematodes. dsRNA-treated nematodes revealed some paralysis and uncoordinated movement in contrast to the regular and sinusoidal movement of the non-treated nematodes. Furthermore, reproduction was reduced in dsRNA-treated nematodes. These results suggest that this protein has a function similar to that of the C. elegans homolog. © 2015 Blackwell Verlag GmbH.


Egas C.,Next Generation Sequencing Unit | Barroso C.,Next Generation Sequencing Unit | Froufe H.J.C.,Next Generation Sequencing Unit | Pacheco J.,Next Generation Sequencing Unit | And 2 more authors.
Standards in Genomic Sciences | Year: 2015

Rubrobacter radiotolerans strain RSPS-4 is a slightly thermophilic member of the phylum "Actinobacteria" isolated from a hot spring in São Pedro do Sul, Portugal. This aerobic and halotolerant bacterium is also extremely resistant to gamma and UV radiation, which are the main reasons for the interest in sequencing its genome. Here, we present the complete genome sequence of strain RSPS-4 as well as its assembly and annotation. We also compare the gene sequence of this organism with that of the type strain of the species R. radiotolerans isolated from a hot spring in Japan. The genome of strain RSPS-4 comprises one circular chromosome of 2,875,491 bp with a G+C content of 66.91%, and 3 circular plasmids of 190,889 bp, 149,806 bp and 51,047 bp, harboring 3,214 predicted protein coding genes, 46 tRNA genes and a single rRNA operon. © 2014 The Author(s).


Figueiredo J.,University of Coimbra | Simoes M.J.,Next Generation Sequencing Unit | Gomes P.,Next Generation Sequencing Unit | Barroso C.,Next Generation Sequencing Unit | And 6 more authors.
PLoS ONE | Year: 2013

The pinewood nematode, Bursaphelenchus xylophilus, is native to North America but it only causes damaging pine wilt disease in those regions of the world where it has been introduced. The accurate detection of the species and its dispersal routes are thus essential to define effective control measures. The main goals of this study were to analyse the genetic diversity among B. xylophilus isolates from different geographic locations and identify single nucleotide polymorphism (SNPs) markers for geographic origin, through a comparative transcriptomic approach. The transcriptomes of seven B. xylophilus isolates, from Continental Portugal (4), China (1), Japan (1) and USA (1), were sequenced in the next generation platform Roche 454. Analysis of effector gene transcripts revealed inter-isolate nucleotide diversity that was validated by Sanger sequencing in the genomic DNA of the seven isolates and eight additional isolates from different geographic locations: Madeira Island (2), China (1), USA (1), Japan (2) and South Korea (2). The analysis identified 136 polymorphic positions in 10 effector transcripts. Pairwise comparison of the 136 SNPs through Neighbor-Joining and the Maximum Likelihood methods and 5-mer frequency analysis with the alignment-independent bilinear multivariate modelling approach correlated the SNPs with the isolates geographic origin. Furthermore, the SNP analysis indicated a closer proximity of the Portuguese isolates to the Korean and Chinese isolates than to the Japanese or American isolates. Each geographic cluster carried exclusive alleles that can be used as SNP markers for B. xylophilus isolate identification. © 2013 Figueiredo et al.


PubMed | Next Generation Sequencing Unit and University of Coimbra
Type: Journal Article | Journal: PloS one | Year: 2014

The pinewood nematode, Bursaphelenchus xylophilus, is native to North America but it only causes damaging pine wilt disease in those regions of the world where it has been introduced. The accurate detection of the species and its dispersal routes are thus essential to define effective control measures. The main goals of this study were to analyse the genetic diversity among B. xylophilus isolates from different geographic locations and identify single nucleotide polymorphism (SNPs) markers for geographic origin, through a comparative transcriptomic approach. The transcriptomes of seven B. xylophilus isolates, from Continental Portugal (4), China (1), Japan (1) and USA (1), were sequenced in the next generation platform Roche 454. Analysis of effector gene transcripts revealed inter-isolate nucleotide diversity that was validated by Sanger sequencing in the genomic DNA of the seven isolates and eight additional isolates from different geographic locations: Madeira Island (2), China (1), USA (1), Japan (2) and South Korea (2). The analysis identified 136 polymorphic positions in 10 effector transcripts. Pairwise comparison of the 136 SNPs through Neighbor-Joining and the Maximum Likelihood methods and 5-mer frequency analysis with the alignment-independent bilinear multivariate modelling approach correlated the SNPs with the isolates geographic origin. Furthermore, the SNP analysis indicated a closer proximity of the Portuguese isolates to the Korean and Chinese isolates than to the Japanese or American isolates. Each geographic cluster carried exclusive alleles that can be used as SNP markers for B. xylophilus isolate identification.


PubMed | Next Generation Sequencing Unit and University of Coimbra
Type: Journal Article | Journal: Standards in genomic sciences | Year: 2014

Rubrobacter radiotolerans strain RSPS-4 is a slightly thermophilic member of the phylum Actinobacteria isolated from a hot spring in So Pedro do Sul, Portugal. This aerobic and halotolerant bacterium is also extremely resistant to gamma and UV radiation, which are the main reasons for the interest in sequencing its genome. Here, we present the complete genome sequence of strain RSPS-4 as well as its assembly and annotation. We also compare the gene sequence of this organism with that of the type strain of the species R. radiotolerans isolated from a hot spring in Japan. The genome of strain RSPS-4 comprises one circular chromosome of 2,875,491 bp with a G+C content of 66.91%, and 3 circular plasmids of 190,889 bp, 149,806 bp and 51,047 bp, harboring 3,214 predicted protein coding genes, 46 tRNA genes and a single rRNA operon.


Simoes M.J.,Next Generation Sequencing Unit | Lobo C.,Association for Innovation and Biomedical Research on Light and Image AIBILI | Lobo C.,Centro Hospitalar iversitario Of Coimbra | Egas C.,Next Generation Sequencing Unit | And 7 more authors.
Ophthalmologica | Year: 2014

Purpose: To explore phenotype-genotype correlations that may contribute to a better understanding of diabetic retinopathy (DR). Procedures: An exploratory association study was performed to identify genetic variants associated with non-proliferative DR (NPDR) in 307 type 2 diabetic patients who were previously stratified into 3 different phenotypes of NPDR progression. The 307 patients were genotyped for 174 single nucleotide polymorphisms of 11 candidate genes (ACE, AGER, AKR1B1, ICAM1, MTHFR, NOS1, NOS3, PPARGC1A, TGFB1, TNF and VEGFA). Results: Significant associations were observed for PPARGC1A rs16874120 with phenotype A (odds ratio, OR = 0.60, 95% confidence interval, CI 0.36-0.99), ICAM1 rs1801714 with phenotype B (OR = 3.32, 95% CI 1.05-10.50) and both PPARGC1A rs10213440 (OR = 2.00, 95% CI 1.07-3.73) and MTHFR rs1801133 (OR = 1.84, 95% CI 1.08-3.11) with phenotype C. Conclusions: Results indicate that specific gene variants in ICAM1, PPARGC1A and MTHFR are associated with different NPDR phenotypes, being likely candidates to explain different disease mechanisms underlying the different phenotypes. This is the first study to show correlations between specific gene variants and NPDR phenotypes, opening new perspectives on DR. © 2014 S. Karger AG, Basel.


PubMed | Next Generation Sequencing Unit
Type: Journal Article | Journal: Ophthalmologica. Journal international d'ophtalmologie. International journal of ophthalmology. Zeitschrift fur Augenheilkunde | Year: 2014

To explore phenotype-genotype correlations that may contribute to a better understanding of diabetic retinopathy (DR).An exploratory association study was performed to identify genetic variants associated with non-proliferative DR (NPDR) in 307 type 2 diabetic patients who were previously stratified into 3 different phenotypes of NPDR progression. The 307 patients were genotyped for 174 single nucleotide polymorphisms of 11 candidate genes (ACE, AGER, AKR1B1, ICAM1, MTHFR, NOS1, NOS3, PPARGC1A, TGFB1, TNF and VEGFA).Significant associations were observed for PPARGC1A rs16874120 with phenotype A (odds ratio, OR = 0.60, 95% confidence interval, CI 0.36-0.99), ICAM1 rs1801714 with phenotype B (OR = 3.32, 95% CI 1.05-10.50) and both PPARGC1A rs10213440 (OR = 2.00, 95% CI 1.07-3.73) and MTHFR rs1801133 (OR = 1.84, 95% CI 1.08-3.11) with phenotype C.RESULTS indicate that specific gene variants in ICAM1, PPARGC1A and MTHFR are associated with different NPDR phenotypes, being likely candidates to explain different disease mechanisms underlying the different phenotypes. This is the first study to show correlations between specific gene variants and NPDR phenotypes, opening new perspectives on DR.

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