Ecological connectivity shapes quasispecies structure of RNA viruses in an Antarctic lake

© 2015 John Wiley & Sons Ltd. RNA viruses exist as complex mixtures of genotypes, known as quasispecies, where the evolution potential resides in the whole community of related genotypes. Quasispecies structure and dynamics have been studied in detail for virus infecting animals and plants but remain unexplored for those infecting micro-organisms in environmental samples. We report the first metagenomic study of RNA viruses in an Antarctic lake (Lake Limnopolar, Livingston Island). Similar to low-latitude aquatic environments, this lake harbours an RNA virome dominated by positive single-strand RNA viruses from the order Picornavirales probably infecting micro-organisms. Antarctic picorna-like virus 1 (APLV1), one of the most abundant viruses in the lake, does not incorporate any mutation in the consensus sequence from 2006 to 2010 and shows stable quasispecies with low-complexity indexes. By contrast, APLV2-APLV3 are detected in the lake water exclusively in summer samples and are major constituents of surrounding cyanobacterial mats. Their quasispecies exhibit low complexity in cyanobacterial mat, but their run-off-mediated transfer to the lake results in a remarkable increase of complexity that may reflect the convergence of different viral quasispecies from the catchment area or replication in a more diverse host community. This is the first example of viral quasispecies from natural aquatic ecosystems and points to ecological connectivity as a modulating factor of quasispecies complexity.Spanish Polar Programme and the Spanish Ministry of Economy and Competitiveness (CTM2008-05134-E/ANT and CTM2009-08644-E).Peer Reviewe

Liquid biopsy by NGS: Differential presence of exons (DPE) is related to metastatic potential in a colon-cancer model in the rat

Differential presence of exons (DPE) is a method of interpretation of exome sequencing, which has been proposed to design a predictive algorithm with clinical value in patients with colorectal cancer (CRC). The goal of the present study was to examine the reproducibility in a rat model of metastatic colon cancer. DHD/K12-TRb cells were injected in syngenic immunocompetent BD-IX rats. Cells were from two stocks with low and normal metastatic potential, and injected into two separate groups of rats. Five to ten weeks after injection, blood samples were taken prior euthanasia and whole exome sequencing performed. Through DPE analysis, we identified a set of exons whose differential presence in plasma allowed us to compare both groups of tumor-bearing animals. A verification test was performed to confirm that the algorithm was able to classify extracted samples into their corresponding groups of origin. The highest mean probability was 0.8954. In conclusion, the DPE analysis in tumor-bearing animals was able to discriminate between different disease status, which fully supports previous results in CRC patients.This studywas funded by two grants from"Instituto de Salud Carlos III", Spain (FIS; refs. PS09/01815 and PI13/01924

ARAMIS: From systematic errors of NGS long reads to accurate assemblies

NGS long-reads sequencing technologies (or third generation) such as Pacific BioSciences (PacBio) have revolutionized the sequencing field over the last decade improving multiple genomic applications like de novo genome assemblies. However, their error rate, mostly involving insertions and deletions (indels), is currently an important concern that requires special attention to be solved. Multiple algorithms are available to fix these sequencing errors using short reads (such as Illumina), although they require long processing times and some errors may persist. Here, we present Accurate long-Reads Assembly correction Method for Indel errorS (ARAMIS), the first NGS long-reads indels correction pipeline that combines several correction software in just one step using accurate short reads. As a proof OF concept, six organisms were selected based on their different GC content, size and genome complexity, and their PacBio-assembled genomes were corrected thoroughly by this pipeline. We found that the presence of systematic sequencing errors in long-reads PacBio sequences affecting homopolymeric regions, and that the type of indel error introduced during PacBio sequencing are related to the GC content of the organism. The lack of knowledge of this fact leads to the existence of numerous published studies where such errors have been found and should be resolved since they may contain incorrect biological information. ARAMIS yields better results with less computational resources needed than other correction tools and gives the possibility of detecting the nature of the found indel errors found and its distribution along the genome. The source code of ARAMIS is available at https://github.com/genomics-ngsCBMSO/ARAMIS.gi

Population structure and genetic variability in wild and farmed Mediterranean populations of gilthead seabream and European seabass inferred from a 60K combined species SNP array

13 Pág.Knowledge of population structure and genetic diversity within and between wild and farmed populations of gilthead sea bream (Sparus aurata) and European seabass (Dicentrarchus labrax) is important to achieve sustainable aquaculture production of these species and to assess the risk of genetic impacts of fish escaped from farms. Previous population genetic studies on these species have been based on a limited number of genetic markers and samples. In this study, these features were assessed using samples from 24 seabream and 25 seabass populations distributed throughout the Mediterranean Sea, and 3 wild seabream Atlantic populations. Samples were genotyped with a newly developed combined species SNP array that includes ~60K SNPs. Data from sequencing pools of individual DNA from the same populations were also used. Different approaches were employed for identifying the extent of population stratification within species. The effective population size (a parameter inversely related to the rate at which genetic variability is lost) was estimated for each population based on linkage disequilibrium. Population structure results revealed a clear differentiation between wild and farmed populations in both species. Wild populations showed a low degree of differentiation, particularly in seabream. Despite this, a slight differentiation was observed between Atlantic and Mediterranean seabream populations and between western and eastern Mediterranean seabass populations. However, farmed populations were quite heterogeneous and showed a high degree of differentiation. Some farmed populations of both species showed a genetic makeup similar to that found in wild populations. In general, the effective population size was large (> 1000) for wild and small (< 100) for farmed populations of both species. About 40% of the seabream and 80% of the seabass farmed populations had estimates of effective population size smaller than 50 highlighting the need of applying measures to control the rate at which genetic variability is lost.This work was supported by the European Commission Horizon 2020 (H2020) Framework Programme through grant agreement no 727315 MedAID project (Mediterranean Aquaculture Integrated Development) and by MCIN/ AEI /10.13039/501100011033 (Project PID2020-114426GB-C2).Peer reviewe

Population structure and genetic variability in wild and farmed Mediterranean populations of gilthead seabream and European seabass inferred from a 60K combined species SNP array

publishedVersio

Mobility of the Native Bacillus subtilis Conjugative Plasmid pLS20 Is Regulated by Intercellular Signaling

Horizontal gene transfer mediated by plasmid conjugation plays a significant role in the evolution of bacterial species, as well as in the dissemination of antibiotic resistance and pathogenicity determinants. Characterization of their regulation is important for gaining insights into these features. Relatively little is known about how conjugation of Gram-positive plasmids is regulated. We have characterized conjugation of the native Bacillus subtilis plasmid pLS20. Contrary to the enterococcal plasmids, conjugation of pLS20 is not activated by recipient-produced pheromones but by pLS20-encoded proteins that regulate expression of the conjugation genes. We show that conjugation is kept in the default >OFF> state and identified the master repressor responsible for this. Activation of the conjugation genes requires relief of repression, which is mediated by an anti-repressor that belongs to the Rap family of proteins. Using both RNA sequencing methodology and genetic approaches, we have determined the regulatory effects of the repressor and anti-repressor on expression of the pLS20 genes. We also show that the activity of the anti-repressor is in turn regulated by an intercellular signaling peptide. Ultimately, this peptide dictates the timing of conjugation. The implications of this regulatory mechanism and comparison with other mobile systems are discussed.Work in the Madrid lab was funded by grant BFU2008-04034/BMC from the Ministry of Science and Innovation of the Spanish Government. PKS is holder of a JaePre fellowship from the Spanish Research Council (CSIC). Work in the Newcastle lab was funded by a Wellcome Trust Investigator Award to Jeff Errington (098374/Z/12/Z)Peer Reviewe

Basolateral sorting and transcytosis define the cu+-regulated translocation of ATP7B to the bile canaliculus

The Cu pump ATP7B plays an irreplaceable role in the elimination of excess Cu by the hepatocyte into the bile. The trafficking and site of action of ATP7B are subjects of controversy. One current proposal is that an increase in intracellular Cu results in the translocation of ATP7B to the lysosomes and excretion of excess Cu through lysosomal-mediated exocytosis at the bile canaliculus. Here, we show that ATP7B is transported from the trans-Golgi network (TGN) to the bile canaliculus by basolateral sorting and endocytosis, and microtubule-mediated transcytosis through the subapical compartment. Trafficking ATP7B is not incorporated into lysosomes, and addition of Cu does not cause relocalization of lysosomes and the appearance of lysosome markers in the bile canaliculus. Our data reveal the pathway of the Cu-mediated transport of ATP7B from the TGN to the bile canaliculus and indicates that the bile canaliculus is the primary site of ATP7B action in the elimination of excess Cu.Instituto de Salud Carlos III [grant number PI11/00922]; the Fundació per Amor a L’Art (2014); and the Fundación Areces (2013).Peer Reviewe

Oxford nanopore sequencing as an alternative to reduced representation bisulphite sequencing for the identification of CpGs of interest in livestock populations

International audienceEpigenetic marks could potentially explain a portion of the phenotypic variance related to traits of interest in animal breeding models. DNA methylation is the most studied epigenetic mark, involving the addition of a methyl group to the fifth carbon of a cytosine, which transforms it into 5-methylcytosine. This mark is commonly associated with inhibiting gene expression without altering the nucleotide sequence, particularly when located within promoter regions. While bisulphite sequencing is the gold standard for detecting methylation marks, new techniques have emerged to address bisulphite's limitations. Some of these limitations include the confounding effects of bisulphite treatment on DNA methylation and C to T polymorphisms, as well as the inability to distinguish between DNA methylation and hydroxylation. In this context, we propose employing Nanopore sequencing to identify methylation marks. We conducted sequencing on six bull semen samples using both Nanopore and reduced representation bisulphite sequencing. CpGs were filtered based on two coverage thresholds (4x and 7x). Our results indicated that Nanopore methylation data exhibited a correlation exceeding 0.95 with bisulphite sequencing data. The correlation decreased to 0.67 when analysing only CpGs with methylation frequencies ranging from 0.1 to 0.9, due to the lack of enough coverage for ONT. We also examined differentially methylated cytosines identified by each sequencing method. The overlap between the two sets of DMCs and the associated genes was limited, as RRBS library preparation predominantly amplifies gene promoter regions, while ONT covers more intergenic regions. Interestingly, both methods highlighted differentially methylated genes and positions linked to quantitative trait loci of significant economic interest in animal breeding, suggesting promising applications for ONT in the field of agri-genomics

Oxford nanopore sequencing as an alternative to reduced representation bisulphite sequencing for the identification of CpGs of interest in livestock populations

12 Pág.Epigenetic marks could potentially explain a portion of the phenotypic variance related to traits of interest in animal breeding models. DNA methylation is the most studied epigenetic mark, involving the addition of a methyl group to the fifth carbon of a cytosine, which transforms it into 5-methylcytosine. This mark is commonly associated with inhibiting gene expression without altering the nucleotide sequence, particularly when located within promoter regions. While bisulphite sequencing is the gold standard for detecting methylation marks, new techniques have emerged to address bisulphite's limitations. Some of these limitations include the confounding effects of bisulphite treatment on DNA methylation and C to T polymorphisms, as well as the inability to distinguish between DNA methylation and hydroxylation. In this context, we propose employing Nanopore sequencing to identify methylation marks. We conducted sequencing on six bull semen samples using both Nanopore and reduced representation bisulphite sequencing. CpGs were filtered based on two coverage thresholds (4x and 7x). Our results indicated that Nanopore methylation data exhibited a correlation exceeding 0.95 with bisulphite sequencing data. The correlation decreased to 0.67 when analysing only CpGs with methylation frequencies ranging from 0.1 to 0.9, due to the lack of enough coverage for ONT. We also examined differentially methylated cytosines identified by each sequencing method. The overlap between the two sets of DMCs and the associated genes was limited, as RRBS library preparation predominantly amplifies gene promoter regions, while ONT covers more intergenic regions. Interestingly, both methods highlighted differentially methylated genes and positions linked to quantitative trait loci of significant economic interest in animal breeding, suggesting promising applications for ONT in the field of agri-genomics.This project has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No 101000226. Open Access funding provided thanks to the CRUE-CSIC agreement with Elsevier.Peer reviewe

Complete and de novo assembly of the Leishmania braziliensis (M2904) genome

Leishmania braziliensis is the etiological agent of American mucosal leishmaniasis, one of the most severe clinical forms of leishmaniasis. Here, we report the assembly of the L. braziliensis (M2904) genome into 35 continuous chromosomes. Also, the annotation of 8395 genes is provided. The public availability of this information will contribute to a better knowledge of this pathogen and help in the search for vaccines and novel drug targets aimed to control the disease caused by this Leishmania species.Ministerio de Economía, Industria y Competitividad SAF2017-86965-R (co-funded with FEDER funds), and ISCIII, project RD16/0027/0008 Red de Enfermedades Tropicales, Subprograma RETICS del Plan Estatal de I+D+I 2013-2016 and co-funded with FEDER funds (grants to BA and JMR), Genomics and NGS Core Facility at the Centro de Biología Molecular Severo Ochoa (CBMSO CSIC-UAM) and the Norwegian Sequencing Centre, a technology platform hosted by the University of Oslo and supported by the “Functional Genomics” and “Infrastructure” programs of the Research Council of Norway and the Southeastern Regional Health Authorities. Institutional grants from the Fundación Ramón Areces and Banco de Santander to the CBMS