RESUMO
OBJECTIVE: To show how to generate a consensus sequence from the information of massive parallel sequences data obtained from routine HIV anti-retroviral resistance studies, and that may be suitable for molecular epidemiology studies. MATERIAL AND METHODS: Paired Sanger (Trugene-Siemens) and next-generation sequencing (NGS) (454 GSJunior-Roche) HIV RT and protease sequences from 62 patients were studied. NGS consensus sequences were generated using Mesquite, using 10%, 15%, and 20% thresholds. Molecular evolutionary genetics analysis (MEGA) was used for phylogenetic studies. RESULTS: At a 10% threshold, NGS-Sanger sequences from 17/62 patients were phylogenetically related, with a median bootstrap-value of 88% (IQR83.5-95.5). Association increased to 36/62 sequences, median bootstrap 94% (IQR85.5-98)], using a 15% threshold. Maximum association was at the 20% threshold, with 61/62 sequences associated, and a median bootstrap value of 99% (IQR98-100). CONCLUSION: A safe method is presented to generate consensus sequences from HIV-NGS data at 20% threshold, which will prove useful for molecular epidemiological studies.
Assuntos
Sequência Consenso , DNA Viral/genética , HIV-1/genética , Sequenciamento de Nucleotídeos em Larga Escala , Epidemiologia Molecular/métodos , Adulto , Fármacos Anti-HIV/farmacologia , Sequência de Bases , Farmacorresistência Viral/genética , HIV-1/classificação , HIV-1/efeitos dos fármacos , Humanos , Filogenia , Análise de Sequência de DNARESUMO
BACKGROUND: The gut microbiota plays a key role in the maintenance of intestinal homeostasis and the development and activation of the host immune system. It has been shown that commensal bacterial species can regulate the expression of host genes. 16S rRNA gene sequencing has shown that the microbiota in inflammatory bowel disease (IBD) is abnormal and characterized by reduced diversity. MicroRNAs (miRNAs) have been explored as biomarkers and therapeutic targets, since they are able to regulate specific genes associated with Crohn's disease (CD). In this work, we aim to investigate the composition of gut microbiota of active treatment-naïve adult CD patients, with miRNA profile from gut microbiota. AIM: To investigate the composition of gut microbiota of active treatment-naïve adult CD patients, with miRNA profile from gut microbiota. METHODS: Patients attending the outpatient clinics at Valme University Hospital without relevant co-morbidities were matched according to age and gender. Faecal samples of new-onset CD patients, free of treatment, and healthy controls were collected. Faecal samples were homogenized, and DNA was amplified by PCR using primers directed to the 16S bacterial rRNA gene. Pyrosequencing was performed using GS-Junior platform. For sequence analysis, MG-RAST server with the database Ribosomal Project was used. MiRNA profile and their relative abundance were analyzed by quantitative PCR. RESULTS: Microbial community was characterized using 16S rRNA gene sequencing in 29 samples (n = 13 CD patients, and n = 16 healthy controls). The mean Shannon diversity was higher in the healthy control population compared to CD group (5.5 vs 3.7). A reduction in Firmicutes and an increase in Bacteroidetes were found. Clostridia class was also significantly reduced in CD. Principal components analysis showed a grouping pattern, identified in most of the subjects in both groups, showing a marked difference between control and CD groups. A functional metabolic study showed that a lower metabolism of carbohydrates (P = 0.000) was found in CD group, while the metabolism of lipids was increased. In CD patients, three miRNAs were induced in affected mucosa: mir-144 (6.2 ± 1.3 fold), mir-519 (21.8 ± 3.1) and mir-211 (2.3 ± 0.4). CONCLUSION: Changes in microbial function in active non-treated CD subjects and three miRNAs in affected vs non-affected mucosa have been found. miRNAs profile may serve as a biomarker.
Assuntos
Bacteroidetes/genética , Doença de Crohn/microbiologia , Fezes/microbiologia , Firmicutes/genética , Microbioma Gastrointestinal/genética , MicroRNAs/isolamento & purificação , Adolescente , Adulto , Bacteroidetes/isolamento & purificação , Biomarcadores/análise , Colonoscopia , Doença de Crohn/diagnóstico , Doença de Crohn/patologia , Feminino , Firmicutes/isolamento & purificação , Perfilação da Expressão Gênica , Humanos , Mucosa Intestinal/diagnóstico por imagem , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Masculino , Metagenoma , MicroRNAs/genética , Pessoa de Meia-Idade , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/isolamento & purificação , Adulto JovemRESUMO
Objetivo: Generar una secuencia consenso a partir de los datos de secuenciación masiva obtenidos en estudios de resistencias a antiretrovirales, que sea representativa de la secuencia Sanger y que sirva para estudios de epidemiología molecular. Material y métodos: En 62 pacientes se obtuvo la secuencia de transcriptasa reversa-proteasa, mediante Sanger (Trugene-Siemens), y NGS (454GSJunior-Roche). Las secuencias consenso NGS se generaron con Mesquite, seleccionando umbrales 10%, 15% y 20%. Para el estudio filogenético se empleó MEGA. Resultados: Utilizando el umbral 10%, 17/62 pacientes presentaron secuencias pareadas NGS-Sanger, con una mediana de bootstrap del 88% (IQR83,5-95,5). La asociación aumenta a 36/62 pacientes y el bootstrap, a 94% (IQR85,5-98), y alcanza el máximo al 20% en 61/62 pacientes, bootstrap 99% (IQR98-100). Conclusión: Mostramos un método seguro para generar secuencias consenso NGS para su uso en estudios de epidemiología molecular procesadas con umbral 20%, de fácil uso y aplicación en los servicios de microbiología clínica (AU)
Objective: To show how to generate a consensus sequence from the information of massive parallel sequences data obtained from routine HIV anti-retroviral resistance studies, and that may be suitable for molecular epidemiology studies. Material and methods: Paired Sanger (Trugene-Siemens) and next-generation sequencing (NGS) (454 GSJunior-Roche) HIV RT and protease sequences from 62 patients were studied. NGS consensus sequences were generated using Mesquite, using 10%, 15%, and 20% thresholds. Molecular evolutionary genetics analysis (MEGA) was used for phylogenetic studies. Results: At a 10% threshold, NGS-Sanger sequences from 17/62 patients were phylogenetically related, with a median bootstrap-value of 88% (IQR83.5-95.5). Association increased to 36/62 sequences, median bootstrap 94% (IQR85.5-98)], using a 15% threshold. Maximum association was at the 20% threshold, with 61/62 sequences associated, and a median bootstrap value of 99% (IQR98-100). Conclusion: A safe method is presented to generate consensus sequences from HIV-NGS data at 20% threshold, which will prove useful for molecular epidemiological studies (AU)