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1.
BMC Genomics ; 18(1): 286, 2017 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-28390408

RESUMO

BACKGROUND: Human endogenous retroviruses (HERVs) have received much attention for their implications in the etiology of many human diseases and their profound effect on evolution. Notably, recent studies have highlighted associations between HERVs expression and cancers (Yu et al., Int J Mol Med 32, 2013), autoimmunity (Balada et al., Int Rev Immunol 29:351-370, 2010) and neurological (Christensen, J Neuroimmune Pharmacol 5:326-335, 2010) conditions. Their repetitive nature makes their study particularly challenging, where expression studies have largely focused on individual loci (De Parseval et al., J Virol 77:10414-10422, 2003) or general trends within families (Forsman et al., J Virol Methods 129:16-30, 2005; Seifarth et al., J Virol 79:341-352, 2005; Pichon et al., Nucleic Acids Res 34:e46, 2006). METHODS: To refine our understanding of HERVs activity, we introduce here a new microarray, HERV-V3. This work was made possible by the careful detection and annotation of genomic HERV/MaLR sequences as well as the development of a new hybridization model, allowing the optimization of probe performances and the control of cross-reactions. RESULTS: HERV-V3 offers an almost complete coverage of HERVs and their ancestors (mammalian apparent LTR-retrotransposons, MaLRs) at the locus level along with four other repertoires (active LINE-1 elements, lncRNA, a selection of 1559 human genes and common infectious viruses). We demonstrate that HERV-V3 analytical performances are comparable with commercial Affymetrix arrays, and that for a selection of tissue/pathological specific loci, the patterns of expression measured on HERV-V3 is consistent with those reported in the literature. CONCLUSIONS: Given its large HERVs/MaLRs coverage and additional repertoires, HERV-V3 opens the door to multiple applications such as enhancers and alternative promoters identification, biomarkers identification as well as the characterization of genes and HERVs/MaLRs modulation caused by viral infection.


Assuntos
Retrovirus Endógenos/genética , Perfilação da Expressão Gênica , Hibridização Genética , Modelos Genéticos , Transcriptoma , Algoritmos , Análise por Conglomerados , Biologia Computacional/métodos , Bases de Dados de Ácidos Nucleicos , Perfilação da Expressão Gênica/métodos , Loci Gênicos , Humanos , Hibridização de Ácido Nucleico , Reprodutibilidade dos Testes , Fluxo de Trabalho
2.
Viruses ; 14(8)2022 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-36016297

RESUMO

Whole-genome sequencing has become an essential tool for real-time genomic surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) worldwide. The handling of raw next-generation sequencing (NGS) data is a major challenge for sequencing laboratories. We developed an easy-to-use web-based application (EPISEQ SARS-CoV-2) to analyse SARS-CoV-2 NGS data generated on common sequencing platforms using a variety of commercially available reagents. This application performs in one click a quality check, a reference-based genome assembly, and the analysis of the generated consensus sequence as to coverage of the reference genome, mutation screening and variant identification according to the up-to-date Nextstrain clade and Pango lineage. In this study, we validated the EPISEQ SARS-CoV-2 pipeline against a reference pipeline and compared the performance of NGS data generated by different sequencing protocols using EPISEQ SARS-CoV-2. We showed a strong agreement in SARS-CoV-2 clade and lineage identification (>99%) and in spike mutation detection (>99%) between EPISEQ SARS-CoV-2 and the reference pipeline. The comparison of several sequencing approaches using EPISEQ SARS-CoV-2 revealed 100% concordance in clade and lineage classification. It also uncovered reagent-related sequencing issues with a potential impact on SARS-CoV-2 mutation reporting. Altogether, EPISEQ SARS-CoV-2 allows an easy, rapid and reliable analysis of raw NGS data to support the sequencing efforts of laboratories with limited bioinformatics capacity and those willing to accelerate genomic surveillance of SARS-CoV-2.


Assuntos
COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Mutação , SARS-CoV-2/genética
3.
Gene ; 411(1-2): 87-93, 2008 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-18281162

RESUMO

Transposable elements, which are major components of most genomes, are known to accumulate in heterochromatic regions in which they have progressively diverged in sequence by mutations and internal deletions and insertions (indels) during the course of evolution. They therefore provide a record of the genomic events that have shaped the genomes, some of which could correspond to speciation events. Using the sequence divergence between the long terminal repeats (LTRs), we estimated the date of the insertion events of the LTR retrotransposon copies embedded within the heterochromatin regions of the Drosophila melanogaster genome. We did not detect traces of any specific waves of mobilization of retrotransposons within heterochromatin, apart from a very recent wave, which corresponds to the numerous LTR retrotransposon copies found in euchromatin.


Assuntos
Drosophila melanogaster/genética , Genoma de Inseto , Heterocromatina , Retroelementos , Sequências Repetidas Terminais , Animais
4.
Front Microbiol ; 9: 511, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29616014

RESUMO

The French National Reference Center for Staphylococci currently uses DNA arrays and spa typing for the initial epidemiological characterization of Staphylococcus aureus strains. We here describe the use of whole-genome sequencing (WGS) to investigate retrospectively four distinct and virulent S. aureus lineages [clonal complexes (CCs): CC1, CC5, CC8, CC30] involved in hospital and community outbreaks or sporadic infections in France. We used a WGS bioinformatics pipeline based on de novo assembly (reference-free approach), single nucleotide polymorphism analysis, and on the inclusion of epidemiological markers. We examined the phylogeographic diversity of the French dominant hospital-acquired CC8-MRSA (methicillin-resistant S. aureus) Lyon clone through WGS analysis which did not demonstrate evidence of large-scale geographic clustering. We analyzed sporadic cases along with two outbreaks of a CC1-MSSA (methicillin-susceptible S. aureus) clone containing the Panton-Valentine leukocidin (PVL) and results showed that two sporadic cases were closely related. We investigated an outbreak of PVL-positive CC30-MSSA in a school environment and were able to reconstruct the transmission history between eight families. We explored different outbreaks among newborns due to the CC5-MRSA Geraldine clone and we found evidence of an unsuspected link between two otherwise distinct outbreaks. Here, WGS provides the resolving power to disprove transmission events indicated by conventional methods (same sequence type, spa type, toxin profile, and antibiotic resistance profile) and, most importantly, WGS can reveal unsuspected transmission events. Therefore, WGS allows to better describe and understand outbreaks and (inter-)national dissemination of S. aureus lineages. Our findings underscore the importance of adding WGS for (inter-)national surveillance of infections caused by virulent clones of S. aureus but also substantiate the fact that technological optimization at the bioinformatics level is still urgently needed for routine use. However, the greatest limitation of WGS analysis is the completeness and the correctness of the reference database being used and the conversion of floods of data into actionable results. The WGS bioinformatics pipeline (EpiSeqTM) we used here can easily generate a uniform database and associated metadata for epidemiological applications.

5.
Int J Antimicrob Agents ; 50(2): 210-218, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28554735

RESUMO

Genetic determinants of antibiotic resistance (AR) have been extensively investigated. High-throughput sequencing allows for the assessment of the relationship between genotype and phenotype. A panel of 672 Pseudomonas aeruginosa strains was analysed, including representatives of globally disseminated multidrug-resistant and extensively drug-resistant clones; genomes and multiple antibiograms were available. This panel was annotated for AR gene presence and polymorphism, defining a resistome in which integrons were included. Integrons were present in >70 distinct cassettes, with In5 being the most prevalent. Some cassettes closely associated with clonal complexes, whereas others spread across the phylogenetic diversity, highlighting the importance of horizontal transfer. A resistome-wide association study (RWAS) was performed for clinically relevant antibiotics by correlating the variability in minimum inhibitory concentration (MIC) values with resistome data. Resistome annotation identified 147 loci associated with AR. These loci consisted mainly of acquired genomic elements and intrinsic genes. The RWAS allowed for correct identification of resistance mechanisms for meropenem, amikacin, levofloxacin and cefepime, and added 46 novel mutations. Among these, 29 were variants of the oprD gene associated with variation in meropenem MIC. Using genomic and MIC data, phenotypic AR was successfully correlated with molecular determinants at the whole-genome sequence level.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Genes Bacterianos , Genótipo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Loci Gênicos , Humanos , Sequências Repetitivas Dispersas , Testes de Sensibilidade Microbiana , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/isolamento & purificação
6.
mBio ; 6(6): e01796-15, 2015 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-26604259

RESUMO

UNLABELLED: Pseudomonas aeruginosa is an antibiotic-refractory pathogen with a large genome and extensive genotypic diversity. Historically, P. aeruginosa has been a major model system for understanding the molecular mechanisms underlying type I clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein (CRISPR-Cas)-based bacterial immune system function. However, little information on the phylogenetic distribution and potential role of these CRISPR-Cas systems in molding the P. aeruginosa accessory genome and antibiotic resistance elements is known. Computational approaches were used to identify and characterize CRISPR-Cas systems within 672 genomes, and in the process, we identified a previously unreported and putatively mobile type I-C P. aeruginosa CRISPR-Cas system. Furthermore, genomes harboring noninhibited type I-F and I-E CRISPR-Cas systems were on average ~300 kb smaller than those without a CRISPR-Cas system. In silico analysis demonstrated that the accessory genome (n = 22,036 genes) harbored the majority of identified CRISPR-Cas targets. We also assembled a global spacer library that aided the identification of difficult-to-characterize mobile genetic elements within next-generation sequencing (NGS) data and allowed CRISPR typing of a majority of P. aeruginosa strains. In summary, our analysis demonstrated that CRISPR-Cas systems play an important role in shaping the accessory genomes of globally distributed P. aeruginosa isolates. IMPORTANCE: P. aeruginosa is both an antibiotic-refractory pathogen and an important model system for type I CRISPR-Cas bacterial immune systems. By combining the genome sequences of 672 newly and previously sequenced genomes, we were able to provide a global view of the phylogenetic distribution, conservation, and potential targets of these systems. This analysis identified a new and putatively mobile P. aeruginosa CRISPR-Cas subtype, characterized the diverse distribution of known CRISPR-inhibiting genes, and provided a potential new use for CRISPR spacer libraries in accessory genome analysis. Our data demonstrated the importance of CRISPR-Cas systems in modulating the accessory genomes of globally distributed strains while also providing substantial data for subsequent genomic and experimental studies in multiple fields. Understanding why certain genotypes of P. aeruginosa are clinically prevalent and adept at horizontally acquiring virulence and antibiotic resistance elements is of major clinical and economic importance.


Assuntos
Antibacterianos/farmacologia , Sistemas CRISPR-Cas , Farmacorresistência Bacteriana , Variação Genética , Filogenia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Biologia Computacional , Genoma Bacteriano , Pseudomonas aeruginosa/classificação , Análise de Sequência de DNA
7.
Leuk Lymphoma ; 55(1): 74-7, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23566160

RESUMO

Granulocyte-colony stimulating factors (G-CSFs) enhance bone marrow (BM) recovery after autologous stem cell transplant (ASCT) in patients with lymphoma and myeloma. Few publications exist that discuss the use of filgrastim biosimilars after ASCT. We conducted a single-center retrospective study in patients with lymphoma and myeloma treated at Brest Hospital to assess the cost reductions related to and the efficiency and safety of filgrastim biosimilars. We identified 65 patients with lymphoma or myeloma treated with filgrastim biosimilars for ASCT and compared 19 parameters of these patients, including BM recovery, side effects, infectious complications and treatment costs, with published historical data on a cohort of 50 patients treated with classic filgrastim. We observed a significant reduction of G-CSF costs in both groups but did not observe a change in total hospitalization costs (representing less than 2% of the costs) between groups. Additionally, we did not observe differences between the two groups in BM recovery, infectious complications, side effects or the other studied parameters. In this retrospective study, the absence of differences between groups after ASCT in lymphoma and myeloma led us to believe that these drugs could be safely and effectively used for such indications without a significant impact on hospitalization costs. A prospective study should be conducted to confirm our results.


Assuntos
Medicamentos Biossimilares/uso terapêutico , Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Transplante de Células-Tronco Hematopoéticas , Adulto , Idoso , Medicamentos Biossimilares/economia , Custos de Medicamentos , Feminino , Filgrastim , Fator Estimulador de Colônias de Granulócitos/economia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Linfoma/terapia , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/terapia , Proteínas Recombinantes/economia , Proteínas Recombinantes/uso terapêutico , Estudos Retrospectivos , Transplante Autólogo , Resultado do Tratamento
8.
J Med Microbiol ; 63(Pt 10): 1311-1315, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25062942

RESUMO

The use of 16S rRNA gene sequences for microbial identification in clinical microbiology is accepted widely, and requires databases and algorithms. We compared a new research database containing curated 16S rRNA gene sequences in combination with the lca (lowest common ancestor) algorithm (RDB-LCA) to a commercially available 16S rDNA Centroid approach. We used 1025 bacterial isolates characterized by biochemistry, matrix-assisted laser desorption/ionization time-of-flight MS and 16S rDNA sequencing. Nearly 80 % of isolates were identified unambiguously at the species level by both classification platforms used. The remaining isolates were mostly identified correctly at the genus level due to the limited resolution of 16S rDNA sequencing. Discrepancies between both 16S rDNA platforms were due to differences in database content and the algorithm used, and could amount to up to 10.5 %. Up to 1.4 % of the analyses were found to be inconclusive. It is important to realize that despite the overall good performance of the pipelines for analysis, some inconclusive results remain that require additional in-depth analysis performed using supplementary methods.


Assuntos
Bactérias/classificação , Técnicas Bacteriológicas/métodos , Biologia Computacional/métodos , Genes de RNAr , Técnicas de Diagnóstico Molecular/métodos , RNA Ribossômico 16S/genética , Análise de Sequência de DNA/métodos , Animais , Bactérias/genética , Bactérias/isolamento & purificação , Infecções Bacterianas/diagnóstico , Infecções Bacterianas/veterinária , Humanos
9.
J Vis Exp ; (81): e50713, 2013 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-24300377

RESUMO

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values(1)​​. 'How to use PSA' remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer(2) or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application(3,4). Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g. PCA3 in prostate cancer(5,6) and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer(7-10). We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1).


Assuntos
Biomarcadores Tumorais/genética , Retrovirus Endógenos/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Neoplasias da Próstata/genética , Neoplasias da Próstata/virologia , Idoso , DNA Complementar/análise , DNA Complementar/genética , DNA de Cadeia Simples/análise , DNA de Cadeia Simples/genética , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , RNA Neoplásico/análise , RNA Neoplásico/genética
10.
PLoS One ; 7(6): e40194, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22761958

RESUMO

Human endogenous retroviruses (HERVs) are spread throughout the genome and their long terminal repeats (LTRs) constitute a wide collection of putative regulatory sequences. Phylogenetic similarities and the profusion of integration sites, two inherent characteristics of transposable elements, make it difficult to study individual locus expression in a large-scale approach, and historically apart from some placental and testis-regulated elements, it was generally accepted that HERVs are silent due to epigenetic control. Herein, we have introduced a generic method aiming to optimally characterize individual loci associated with 25-mer probes by minimizing cross-hybridization risks. We therefore set up a microarray dedicated to a collection of 5,573 HERVs that can reasonably be assigned to a unique genomic position. We obtained a first view of the HERV transcriptome by using a composite panel of 40 normal and 39 tumor samples. The experiment showed that almost one third of the HERV repertoire is indeed transcribed. The HERV transcriptome follows tropism rules, is sensitive to the state of differentiation and, unexpectedly, seems not to correlate with the age of the HERV families. The probeset definition within the U3 and U5 regions was used to assign a function to some LTRs (i.e. promoter or polyA) and revealed that (i) autonomous active LTRs are broadly subjected to operational determinism (ii) the cellular gene density is substantially higher in the surrounding environment of active LTRs compared to silent LTRs and (iii) the configuration of neighboring cellular genes differs between active and silent LTRs, showing an approximately 8 kb zone upstream of promoter LTRs characterized by a drastic reduction in sense cellular genes. These gathered observations are discussed in terms of virus/host adaptive strategies, and together with the methods and tools developed for this purpose, this work paves the way for further HERV transcriptome projects.


Assuntos
Retrovirus Endógenos/genética , Análise de Sequência com Séries de Oligonucleotídeos , Transcriptoma
11.
Mol Biol Evol ; 22(3): 747-57, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15574808

RESUMO

There are no doubts that transposable elements (TEs) have greatly influenced genomes evolution. They have, however, evolved in different ways throughout mammals, plants, and invertebrates. In mammals they have been shown to be widely present but with low transposition activity; in plants they are responsible for large increases in genome size. In Drosophila, despite their low amount, transposition seems to be higher. Therefore, to understand how these elements have evolved in different genomes and how host genomes have proposed to go around them, are major questions on genome evolution. We analyzed sequences of the retrotransposable elements 412 in natural populations of the Drosophila simulans and D. melanogaster species that greatly differ in their amount of TEs. We identified new subfamilies of this element that were the result of mutation or insertion-deletion process, but also of interfamily recombinations. These new elements were well conserved in the D. simulans natural populations. The new regulatory regions produced by recombination could give rise to new elements able to overcome host control of transposition and, thus, become potential genome invaders.


Assuntos
Genoma , Recombinação Genética , Sequências Reguladoras de Ácido Nucleico/genética , Retroelementos/genética , Animais , Sequência de Bases , Drosophila melanogaster , Evolução Molecular , Genética Populacional , Humanos , Dados de Sequência Molecular , Mutagênese Insercional , Deleção de Sequência
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