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1.
Clin Microbiol Infect ; 27(1): 130.e5-130.e8, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-33007476

RESUMEN

OBJECTIVES: Investigation whether in depth characterization of virus variant patterns can be used for epidemiological analysis of the first severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection clusters in Hamburg, Germany. METHODS: Metagenomic RNA-sequencing and amplicon-sequencing and subsequent variant calling in 25 respiratory samples from SARS-CoV-2 infected patients involved in the earliest infection clusters in Hamburg. RESULTS: Amplikon sequencing and cluster analyses of these SARS-CoV-2 sequences allowed the identification of the first infection cluster and five non-related infection clusters occurring at the beginning of the viral entry of SARS-CoV-2 in the Hamburg metropolitan region. Viral genomics together with epidemiological analyses revealed that the index patient acquired the infection in northern Italy and transmitted it to two out of 134 contacts. Single nucleotide polymorphisms clearly distinguished the virus variants of the index and other clusters and allowed us to track in which sequences worldwide these mutations were first described. Minor variant analyses identified the transmission of intra-host variants in the index cluster and household clusters. CONCLUSIONS: SARS-CoV-2 variant tracing allows the identification of infection clusters and the follow up of infection chains occurring in the population. Furthermore, the follow up of minor viral variants in infection clusters can provide further resolution on transmission events indistinguishable at a consensus sequence level.


Asunto(s)
/genética , /transmisión , Genoma Viral , Pandemias/prevención & control , /genética , Adulto , /biosíntesis , Trazado de Contacto/estadística & datos numéricos , Evolución Molecular , Femenino , Alemania/epidemiología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Italia/epidemiología , Masculino , Familia de Multigenes , Filogenia , Polimorfismo de Nucleótido Simple , /patogenicidad , Viaje
2.
J Clin Pathol ; 74(1): 19-24, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-32385139

RESUMEN

BACKGROUND: Peritoneal metastasis from pancreatic cancer (PM-PC) may be treated with repeated pressurised intraperitoneal aerosol chemotherapy (PIPAC). Utility of next-generation sequencing (NGS) to detect cancer-related mutations in peritoneal quadrant biopsies (QBs) and peritoneal fluid (PF) after systemic and PIPAC treatment has not been evaluated. Around 90% of pancreatic cancers (PCs) harbour a KRAS mutation, making PC ideal for the evaluation of this aspect. AIMS: Evaluation of PM-PC in terms of (1) histological response to PIPAC using Peritoneal Regression Grading Score (PRGS), (2) clinical characteristics and (3) frequency of mutations in QBs and PF before and after PIPAC. METHODS: Peritoneal QBs and PF were obtained prior to each PIPAC. NGS for 22 cancer-related genes was performed on primary tumours, QBs and PFs. Response was assessed by the four-tiered PRGS. RESULTS: Sixteen patients treated with a median of three PIPAC procedures were included. The mean PRGS was reduced from 1.91 to 1.58 (p=0.02). Fifty-seven specimens (13 primary tumours, 2 metastatic lymph nodes, 16 PFs and 26 QB sets) were analysed with NGS. KRAS mutation was found in 14/16 patients (87.50%) and in QBs, primary tumours and PF in 8/12 (66.67%), 8/13 (61.53%) and 6/9 (66.67%). The median overall survival was 9.9 months (SE 1.5, 95% CI 4.9 to 13.9). CONCLUSION: PIPAC induces histological response in the majority of patients with PM-PC. KRAS mutation can be found in PM-PC after PIPAC at a frequency similar to the primaries. NGS may be used to detect predictive mutations in PM-PC of various origins, also when only post-PIPAC QBs or PFs are available.


Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Neoplasias Pancreáticas/genética , Neoplasias Peritoneales/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Anciano , Líquido Ascítico/patología , Biopsia , Cisplatino/administración & dosificación , Doxorrubicina/administración & dosificación , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inyecciones Intraperitoneales , Masculino , Persona de Mediana Edad , Mutación , Metástasis de la Neoplasia , Oncogenes , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Neoplasias Peritoneales/tratamiento farmacológico , Neoplasias Peritoneales/secundario , Peritoneo/patología , Análisis de Secuencia de ADN
3.
J Clin Pathol ; 74(1): 43-47, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-32467321

RESUMEN

AIMS: Osimertinib is a third-generation EGFR (epidermal growth factor receptor) tyrosine kinase inhibitor that is effective in non-small cell lung cancer (NSCLC) harbouring the EGFR T790M mutation. The Idylla EGFR Mutation Test is a rapid cartridge-based method for detecting T790M and other EGFR mutations. However, false negative T790M results have been reported, and the sensitivity of the assay for this mutation is uncertain. METHODS: Eighty NSCLC samples were tested by both Idylla and a next-generation sequencing (NGS) assay; 46 were from patients at disease progression, and 24 of these had known T790M mutations. Droplet digital PCR (ddPCR) was used to confirm NGS findings in samples with the T790M mutation. RESULTS: Of 19 samples with T790M variant allele frequencies (VAF) higher than the stated 5% limit of detection, 14 were detected by Idylla (sensitivity 74%, 95% CI 49% to 90%). Where sufficient sample remained, ddPCR was consistent with NGS findings in all samples. False negative T790M results were associated with higher EGFR control Cq values (median 22.8 vs 19.8), presence of the EGFR Q787Q polymorphism in cis (80% vs 44%) and presence of an invalid T790M amplification curve. An EGFR exon 19 indel with VAF >5% was also not detected by the Idylla assay in two samples. CONCLUSIONS: The Idylla EGFR Mutation Test has reduced sensitivity for the T790M mutation compared with NGS and ddPCR methods. The presence of an invalid T790M amplification curve may indicate a possible false negative result that warrants further testing by an orthogonal method.


Asunto(s)
Acrilamidas/farmacología , Compuestos de Anilina/farmacología , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/genética , Inhibidores de Proteínas Quinasas/farmacología , Alelos , Sustitución de Aminoácidos , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Variación Genética , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/tratamiento farmacológico , Mutación , Sensibilidad y Especificidad , Análisis de Secuencia de ADN
4.
Food Chem ; 339: 128099, 2021 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-33152884

RESUMEN

Microbial diversity in kefir grains is responsible for the production of goat milk kefir with unique peptides composition and volatile profile. High-throughput sequencing technique was used to analyze bacterial and fungal diversity of three different kefir grains which were originated from China, Europe Germany and United States. Peptides and volatile profile in goat milk kefir were determined by proteomic platform and Gas Chromatography-Ion Mobility Spectrometry, respectively. Clustering analysis indicated that the different content of Lactobacillus genera in different kefir grains was highly associated with the proteolytic ability in goat milk kefir. Contents of volatile compounds in goat milk kefir were also correlated to the bacteria and fungi in kefir grains (especially for Lactobacillus spp. and Saccharomyces spp.). The innovation of this study was to find a new way in exploration of the correlation of microbiota in kefir grains with the proteolytic ability and volatile profile of goat milk kefir.


Asunto(s)
Kéfir/análisis , Kéfir/microbiología , Proteínas de la Leche/análisis , Compuestos Orgánicos Volátiles/análisis , Animales , Bacterias/genética , China , Europa (Continente) , Microbiología de Alimentos , Hongos/genética , Alemania , Cabras , Secuenciación de Nucleótidos de Alto Rendimiento , Lactobacillus/genética , Microbiota/genética , Leche/microbiología , Proteínas de la Leche/metabolismo , Proteolisis , Saccharomyces/genética , Estados Unidos
5.
Gene ; 765: 145091, 2021 Jan 10.
Artículo en Inglés | MEDLINE | ID: mdl-32898604

RESUMEN

Sequencing transposon mutant libraries have been pivotal in annotating essential and non-essential genes in bacteria. This is particularly very helpful in the case of Mycobacterium tuberculosis with a large part of its genome without known function. It is not known whether there are any variations in the essentiality states as a function of optimal growth in the absence of any selection pressure. We here grow a high-density mutant library of M. tuberculosis through serial cultures and monitor the temporal fluctuations in insertion frequencies across all TA dinucleotides in the genome. Genes that cause morphological and physiological heterogeneity or enable metabolic bypass were found to gradually lose insertions, while genes comprising the toxin-antitoxin systems were found to get enriched with insertions during growth in nutrient replete conditions. High levels of fluctuations were observed in genes involved in cell wall and cell processes, intermediary metabolism, and genes involved in virulence, suggesting new modes of adaptation undertaken by the mutants. We also report the essentiality status of several newly annotated genetic features.


Asunto(s)
Elementos Transponibles de ADN/genética , Genes Esenciales/genética , Mycobacterium tuberculosis/genética , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Mutagénesis Insercional/genética , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidad , Sistemas Toxina-Antitoxina/genética , Virulencia/genética
6.
Virus Res ; 291: 198201, 2021 01 02.
Artículo en Inglés | MEDLINE | ID: mdl-33080244

RESUMEN

Here a bioinformatic pipeline VVV has been developed to analyse viral populations in a given sample from Next Generation Sequencing (NGS) data. To date, handling large amounts of data from NGS requires the expertise of bioinformaticians, both for data processing and result analysis. Consequently, VVV was designed to help non-bioinformaticians to perform these tasks. By providing only the NGS data file, the developed pipeline generated consensus sequences and determined the composition of the viral population for an avian Metapneumovirus (AMPV) and three different animal coronaviruses (Porcine Epidemic Diarrhea Virus (PEDV), Turkey Coronavirus (TCoV) and Infectious Bronchitis Virus (IBV)). In all cases, the pipeline produced viral consensus genomes corresponding to known consensus sequence and made it possible to highlight the presence of viral genetic variants through a single graphic representation. The method was validated by comparing the viral populations of an AMPV field sample, and of a copy of this virus produced from a DNA clone. VVV demonstrated that the cloned virus population was homogeneous (as designed) at position 2934 where the wild-type virus demonstrated two variant populations at a ratio of almost 50:50. A total of 18, 10, 3 and 28, viral genetic variants were detected for AMPV, PEDV, TCoV and IBV respectively. The simplicity of this pipeline makes the study of viral genetic variants more accessible to a wide variety of biologists, which should ultimately increase the rate of understanding of the mechanisms of viral genetic evolution.


Asunto(s)
Biología Computacional/instrumentación , Variación Genética , Genoma Viral , Animales , Gráficos por Computador , Coronavirus/genética , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Metapneumovirus/genética , ARN Viral , Recombinación Genética
7.
Gan To Kagaku Ryoho ; 47(12): 1645-1652, 2020 Dec.
Artículo en Japonés | MEDLINE | ID: mdl-33342975

RESUMEN

Cancer precision medicine has become widespread in the world. In Japan, genomic profiling tumor tissues using next generation sequencing(NGS)has been reimbursed to enable simultaneous measurement of multiple biomarkers and genomic abnormalities in clinical practice. However, NGS analysis of tumor tissue has several problems, including long turnaround time, and difficulty in capturing heterogeneity and longitudinal genotyping. Liquid biopsy, an advanced technique that has been developed in recent years, can assess the status of tumors using samples of body fluids, such as blood and urine, without the use of tumor tissue. In particular, circulating tumor DNA(ctDNA), which is released from tumor cells into the blood by apoptosis and necrosis, can be used to select molecularly targeted therapies, monitor therapeutic efficacy, and determine risk of recurrence and select for adjuvant chemotherapy by assessment of minimal residual disease(MRD). In this review, we outline the usefulness, disadvantages and future perspectives of ctDNA analysis.


Asunto(s)
ADN Tumoral Circulante , Biomarcadores de Tumor/genética , ADN Tumoral Circulante/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Japón , Mutación , Recurrencia Local de Neoplasia , Medicina de Precisión
8.
Zhongguo Zhong Yao Za Zhi ; 45(21): 5169-5176, 2020 Nov.
Artículo en Chino | MEDLINE | ID: mdl-33350232

RESUMEN

In order to study the transcriptional differences of Citrus medica var. sarcodactylis at different developmental stages, we explored the genes regulating the biosynthesis of the effective components. In this study, Illumina Hiseq 4 000 high-throughput sequencing technology was used to sequence the transcriptome of C. medica var. sarcodactylis at different developmental stages, 121 235 unigenes were obtained with an average length of 2 434 bp, 3 379 different genes were obtained using DESeq screening, which mainly connected to biological processes such as signal transmission, biological regulation, and metabolic processes, and enriched in metabolic pathways such as starch, sucrose metabolism, phenylpropanoid biosynthesis, and flavonoid biosynthesis. Further dynamic comparison of biosynthesis related genes of active ingredients: the expression levels of PAL, CHI, CYP75B1, ZDS, 4CL and FLS gradually increased as the fruit turned from green to yellow; the expressions of COMT, F3H and CYP73A increased at first and then decreased; CCR, HCT and HRP were down-regulated whereas up-regulated. This study provides references for further excavation of key genes in the biosynthesis of active components, as well as biopathway analysis of active components for C. medica var. sarcodactylis.


Asunto(s)
Citrus , Citrus/genética , Biología Computacional , Frutas/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Secuenciación de Nucleótidos de Alto Rendimiento , Transcriptoma
9.
BMC Bioinformatics ; 21(Suppl 9): 523, 2020 Dec 03.
Artículo en Inglés | MEDLINE | ID: mdl-33272199

RESUMEN

Cancer is one of the leading causes of morbidity and mortality in the globe. Microbiological infections account for up to 20% of the total global cancer burden. The human microbiota within each organ system is distinct, and their compositional variation and interactions with the human host have been known to attribute detrimental and beneficial effects on tumor progression. With the advent of next generation sequencing (NGS) technologies, data generated from NGS is being used for pathogen detection in cancer. Numerous bioinformatics computational frameworks have been developed to study viral information from host-sequencing data and can be adapted to bacterial studies. This review highlights existing popular computational frameworks that utilize NGS data as input to decipher microbial composition, which output can predict functional compositional differences with clinically relevant applicability in the development of treatment and prevention strategies.


Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento , Microbiota/genética , Neoplasias/microbiología , Especificidad de Órganos/genética , Biología Computacional , Humanos
10.
BMJ ; 371: m4050, 2020 12 07.
Artículo en Inglés | MEDLINE | ID: mdl-33288500

RESUMEN

Cancers of unknown primary (CUPs) are histologically confirmed, metastatic malignancies with a primary tumor site that is unidentifiable on the basis of standard evaluation and imaging studies. CUP comprises 2-5% of all diagnosed cancers worldwide and is characterized by early and aggressive metastasis. Current standard evaluation of CUP requires histopathologic evaluation and identification of favorable risk subtypes that can be more definitively treated or have superior outcomes. Current standard treatment of the unfavorable risk subtype requires assessment of prognosis and consideration of empiric chemotherapy. The use of molecular tissue of origin tests to identify the likely primary tumor site has been extensively studied, and here we review the rationale and the evidence for and against the use of such tests in the assessment of CUPs. The expanding use of next generation sequencing in advanced cancers offers the potential to identify a subgroup of patients who have actionable genomic aberrations and may allow for further personalization of therapy.


Asunto(s)
Biomarcadores de Tumor/metabolismo , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Técnicas de Diagnóstico Molecular , Neoplasias Primarias Desconocidas/diagnóstico , Quinasa de Linfoma Anaplásico/genética , Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biopsia , ADN Tumoral Circulante/genética , Genes erbB-1 , Genes erbB-2 , Humanos , Ganglios Linfáticos/patología , Mamografía , Terapia Molecular Dirigida , Mutación , Neoplasias Primarias Desconocidas/tratamiento farmacológico , Neoplasias Primarias Desconocidas/epidemiología , Neoplasias Primarias Desconocidas/genética , Sangre Oculta , Examen Físico , Rendimiento Físico Funcional , Tomografía Computarizada por Tomografía de Emisión de Positrones , Proteínas Proto-Oncogénicas B-raf/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Medición de Riesgo , Tomografía Computarizada por Rayos X
11.
Zhonghua Shao Shang Za Zhi ; 36(12): 1159-1166, 2020 Dec 20.
Artículo en Chino | MEDLINE | ID: mdl-33379852

RESUMEN

Objective: To analyze the dynamic change rule of gut microbiota in patients with extremely severe burns using 16S ribosomal RNA (rRNA) high-throughput sequencing technology. Methods: Five patients with extremely severe burns who were admitted to Hwa Mei Hospital of University of Chinese Academy of Sciences from February to June 2017 and conformed to the inclusion criteria were included in the prospective observational study. All patients were males with age of 32-48 years. Fecal samples were collected in the shock stage (within 3 days after injury), early stage of acute infection (4-14 d after injury), middle stage of acute infection (15-28 d after injury), late stage of acute infection (from 29 d after injury to 1 week before discharge) and within 1 week before discharge. The number of samples was 5 in each stage. The fecal pH value was measured using a pH meter. High-throughput sequencing technology was applied for sequencing of 16S rRNA V3 and V4 regions of fecal samples. QIIME software was used to analyze the number of operational taxonomic units (OTUs), α diversity (Chao1 index and Shannon index), and the relative abundance of gut microbiota at the phylum and family levels. Unweighted pair group method with arithmetic mean clustering method was used to analyze the ß diversity of gut microbiota, and Tax4Fun was used to predict functional changes of gut microbiota. Data were statistically analyzed with one-way analysis of variance for repeated measurement, Bonferroni method, Wilcoxon rank sum test for paired samples, and Bonferroni correction. Results: (1) The pH value of feces in the early and middle stages of acute infection in patients with extremely severe burns in this group was 7.40±0.45 and 7.56±0.45 respectively, which were significantly higher than 6.68±0.36 in the shock stage (P<0.05 or P<0.01). (2) A total of 2 333 584 efficient and high-quality sequences were obtained, and the length of the sequences was about 415 bp. A total of 1 209 OTUs were obtained. The sequencing coverage of all samples was over 99.0%. The number of OTUs and Chao1 index in the early, middle, and late stages of acute infection in patients with extremely severe burns in this group were significantly lower than those in the shock stage (Z=2.023, P<0.05). The number of OTUs and Chao1 index within 1 week before discharge were significantly higher than those in the early, middle, and late stages of acute infection, and Shannon index within 1 week before discharge was significantly higher than that in the early and middle stages of acute infection (Z=2.023, P<0.05). (3) The structure of gut microbiota in the shock stage in patients with extremely severe burns in this group was highly similar to that within 1 week before discharge, and lowly similar to that in the early, middle, and late stages of acute infection. The analysis of individual sample showed that the clustering rule of most of the samples was in accordance with that of the staged samples. The weighted Unifrac distance of gut microbiota in the shock stage was significantly shorter than that in the early, middle, and late stages of acute infection (Z=3.326, 2.570, 2.690, P<0.05 or P<0.01), while the weighted Unifrac distance of gut microbiota in the other stages was similar. (4) At the phylum level, compared with that in the shock stage, the relative abundance of Firmicutes was decreased in the early, middle, and late stages of acute infection, while the relative abundance of Bacteroidetes and Proteobacteria increased. However, the relative abundance of the above three phyla within 1 week before discharge was similar to that in the shock stage. At the family level, the top five dominant bacteria in relative abundance in different stages after injury were quite different. The relative abundance of dominant five family bacteria in the shock stage was decreased in the early, middle, and late stages of acute infection. The relative abundance of non-dominant bacteria such as Enterobacteriaceae, Streptococcaceae, and Bacteroidaceae in the shock stage increased significantly in the early, middle, and late stages of acute infection, which became new dominant families in these stages. The relative abundance of some acid-producing bacteria within 1 week before discharge resumed to the similar level in the shock stage. (5) Functions such as some amino acid metabolism, glycolysis and gluconeogenesis, and pyruvate metabolism of gut microbiota were obviously weaker in the early and middle stages of acute infection than those in the shock stage. Functions such as some amino acid metabolism and carbohydrate metabolism of gut microbiota were significantly enhanced in the late stage of acute infection compared with that in the shock stage. The distributions of functional genes in gut microbiota were similar between the shock stage and within 1 week before discharge. Conclusions: The internal environment and gut microbial compositions in extremely severe burned patients change significantly in the early and middle stages of acute infection. The pH value increases, the bacterial species and diversity decrease, especially the relative abundance of acid-produced bacteria is significantly reduced, which gradually recover with the improvement of the patient's condition. The pH value and the changes of Proteobacteria and acid-producing bacteria could be considered as suitable parameters for reflecting the disorder level of gut microbiota in patients with extremely severe burns.


Asunto(s)
Quemaduras , Microbioma Gastrointestinal , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , ARN Ribosómico 16S/genética , Tecnología
12.
Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi ; 32(6): 618-622, 2020 Oct 15.
Artículo en Chino | MEDLINE | ID: mdl-33325197

RESUMEN

OBJECTIVE: To obtain the transcriptome data of Tyrophagus putrescentiae, so as to provide insights into the subsequent functional studies. METHODS: The mixture of male and female T. putrescentiae was sequenced using the Illumina HiSeqTM 2000 high-throughput sequencing platform. Unigenes were obtained after assembling the sequencing data using the Trinity software and compared with the protein sequences in the RefSeq non-redundant protein sequence (NR) database, nucleotide sequence (NT) database, Swiss-Prot database, Kyoto encyclopedia of genes and genomes (KEGG) database and clusters of orthologous groups (COG) database, and the function of the Unigenes was annotated. In addition, the coding DNA sequences (CDS) were predicted through alignment of the Unigenes in NR and Swiss-Prot protein databases. The SSR loci were identified by analysis of the Unigenes in T. putrescentiae with the MISA software, and the SNPs were detected using the SOAPsnp technique. RESULTS: A total of 4.67 GB high-quality data were obtained from raw sequencing data. A total of 51 271 Unigenes were obtained after assembling the sequencing data, with a total length of 41 848 995 nucleotide (nt) and a mean length of 816 nt. A total of 29 053 annotated Unigenes were obtained following comparisons with the public protein databases, and 27 443 CDS were predicted. In addition, there were 23 092 SSR loci and 148 027 SNPs identified. CONCLUSIONS: The database of T. putrescentiae transcriptome is created by sequencing, and a large number of T. putrescentiae transcripts are obtained, which provides a basis for the subsequent functional studies of allergy-related genes.


Asunto(s)
Acaridae/genética , Biología Computacional , Transcriptoma , Animales , Secuencia de Bases , Bases de Datos de Ácidos Nucleicos , Femenino , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Masculino , Repeticiones de Microsatélite , Anotación de Secuencia Molecular , Polimorfismo de Nucleótido Simple
13.
Sci Total Environ ; 749: 142262, 2020 Dec 20.
Artículo en Inglés | MEDLINE | ID: mdl-33370926

RESUMEN

The ecological restoration of ecosystem services and biodiversity is a key intervention used to reverse the impacts of anthropogenic activities such as mining. Assessment of the performance of restoration against completion criteria relies on biodiversity monitoring. However, monitoring usually overlooks soil microbial communities (SMC), despite increased awareness of their pivotal role in many ecological functions. Recent advances in cost, scalability and technology has led to DNA sequencing being considered as a cost-effective biological monitoring tool, particularly for otherwise difficult to survey groups such as microbes. However, such approaches for monitoring complex restoration sites such as post-mined landscapes have not yet been tested. Here we examine bacterial and fungal communities across chronosequences of mine site restoration at three locations in Western Australia to determine if there are consistent changes in SMC diversity, community composition and functional capacity. Although we detected directional changes in community composition indicative of microbial recovery, these were inconsistent between locations and microbial taxa (bacteria or fungi). Assessing functional diversity provided greater understanding of changes in site conditions and microbial recovery than could be determined through assessment of community composition alone. These results demonstrate that high-throughput amplicon sequencing of environmental DNA (eDNA) is an effective approach for monitoring the complex changes in SMC following restoration. Future monitoring of mine site restoration using eDNA should consider archiving samples to provide improved understanding of changes in communities over time. Expansion to include other biological groups (e.g. soil fauna) and substrates would also provide a more holistic understanding of biodiversity recovery.


Asunto(s)
Microbiota , Suelo , Biodiversidad , Ecosistema , Secuenciación de Nucleótidos de Alto Rendimiento , Microbiología del Suelo , Australia Occidental
14.
BMC Bioinformatics ; 21(Suppl 21): 570, 2020 Dec 28.
Artículo en Inglés | MEDLINE | ID: mdl-33371875

RESUMEN

BACKGROUND: Genome assembly is fundamental for de novo genome analysis. Hybrid assembly, utilizing various sequencing technologies increases both contiguity and accuracy. While such approaches require extra costly sequencing efforts, the information provided millions of existed whole-genome sequencing data have not been fully utilized to resolve the task of scaffolding. Genetic recombination patterns in population data indicate non-random association among alleles at different loci, can provide physical distance signals to guide scaffolding. RESULTS: In this paper, we propose LDscaff for draft genome assembly incorporating linkage disequilibrium information in population data. We evaluated the performance of our method with both simulated data and real data. We simulated scaffolds by splitting the pig reference genome and reassembled them. Gaps between scaffolds were introduced ranging from 0 to 100 KB. The genome misassembly rate is 2.43% when there is no gap. Then we implemented our method to refine the Giant Panda genome and the donkey genome, which are purely assembled by NGS data. After LDscaff treatment, the resulting Panda assembly has scaffold N50 of 3.6 MB, 2.5 times larger than the original N50 (1.3 MB). The re-assembled donkey assembly has an improved N50 length of 32.1 MB from 23.8 MB. CONCLUSIONS: Our method effectively improves the assemblies with existed re-sequencing data, and is an potential alternative to the existing assemblers required for the collection of new data.


Asunto(s)
Desequilibrio de Ligamiento , Secuenciación Completa del Genoma/métodos , Alelos , Animales , Secuenciación de Nucleótidos de Alto Rendimiento , Porcinos
15.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 37(12): 1371-1375, 2020 Dec 10.
Artículo en Chino | MEDLINE | ID: mdl-33306825

RESUMEN

OBJECTIVE: To explore the genetic basis for an infant with neonatal diabetes (NDM) and multiple malformations. METHODS: Genetic variants were detected by next generation sequencing (NGS). Suspected variant was verified by Sanger sequencing. RESULTS: A de novo heterozygous variant, c.1454_1455del(p.K485Rfs), was detected in exon 5 of the GATA6 gene. The variant was undetected in his parents and unreported previously. Bioinformatic analysis predicted the variant to be pathogenic. CONCLUSION: The heterozygous variant of c.1454_1455del(p.K485Rfs) of the GATA6 gene probably underlies the disease in this child. Genetic testing can facilitate diagnosis and genetic counseling for NDM.


Asunto(s)
Anomalías Múltiples , Diabetes Mellitus , Eliminación de Secuencia , Adulto , Diabetes Mellitus/genética , Femenino , Pruebas Genéticas , Heterocigoto , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Recién Nacido , Masculino , Eliminación de Secuencia/genética
16.
BMC Med Genet ; 21(1): 240, 2020 12 12.
Artículo en Inglés | MEDLINE | ID: mdl-33308164

RESUMEN

BACKGROUND: In Morocco, consanguinity rate is very high; which lead to an increase in the birth prevalence of infants with autosomal recessive disorders. Previously, it was difficult to diagnose rare autosomal recessive diseases. Next Generation Sequencing (NGS) techniques have considerably improved clinical diagnostics. A genetic diagnosis showing biallelic causative mutations is the requirement for targeted carrier testing in parents, prenatal and preimplantation genetic diagnosis in further pregnancies, and also for targeted premarital testing in future couples at risk of producing affected children by a known autosomal recessive disease. METHODS: In this report, we present our strategy to advise a future couple of first cousins, whose descendants would risk cystinosis; an autosomal recessive lysosomal disease caused by mutations in the CTNS gene. Indeed, our future husband's sister is clinically and biochemically diagnosed with cystinosis in early childhood. First, we opted to identify the patient's CTNS gene abnormality by using (NGS), then we searched for heterozygosity in the couple's DNA, which allows us to predict the exact risk of this familial disease in the future couple's offspring. RESULTS: We have shown that the future husband, brother of the patient is heterozygous for the familial mutation. On the other hand, his future wife did not inherit the familial mutation. Therefore, genetic counseling was reassuring for the risk of familial cystinosis in this couple's offspring. CONCLUSIONS: We report in this study, one of the major applications of (NGS), an effective tool to improve clinical diagnosis and to provide the possibility of targeted premarital carrier testing in couples at risk.


Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros/genética , Consanguinidad , Cistinosis/genética , Asesoramiento Genético , Mutación , Adulto , Sistemas de Transporte de Aminoácidos Neutros/deficiencia , Cistinosis/diagnóstico , Cistinosis/patología , Femenino , Expresión Génica , Pruebas Genéticas , Heterocigoto , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Marruecos , Linaje , Riesgo
18.
Eur Rev Med Pharmacol Sci ; 24(22): 11914-11918, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-33275263

RESUMEN

OBJECTIVE: Herein we report clinical and virological data in a patient with COVID-19 infection and a prior history of kidney transplantation who had a good clinical recovery despite systemic infection. PATIENTS AND METHODS: Reverse transcriptase quantitative PCR analysis for the RdRp, N and E target genes detected viral RNA in different types of biological specimens. Whole viral genome sequences were obtained and analyzed from respiratory tract, feces and blood. RESULTS: Viral sequences showed high (~99.9%) homology with the Wuhan seafood market pneumonia virus. Phylogenetic analysis assigned of the SARS-CoV-2 strains to clade G. A rare variant in the orf1ab gene was present in both sequences, while a missense variant was detected only in viral RNA from stool. CONCLUSIONS: The evolution of the COVID-19 systemic infection in the patient presented here was favorable to the hypothesis that immunosuppressive therapy in organ transplant recipients might be involved in viral dissemination. A missense mutation was present in only one specimen from the same patient implying the occurrence of a mutational event in viral RNA, which is suggestive for the presence of an active virus, even though viral isolation is necessary to demonstrate infectivity.


Asunto(s)
/virología , Heces/virología , Trasplante de Riñón , Nasofaringe/virología , ARN Viral/genética , Proteínas Virales/genética , Heces/química , Femenino , Rechazo de Injerto/prevención & control , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunosupresores/uso terapéutico , Persona de Mediana Edad , Mutación Missense/genética , Nasofaringe/química , Filogenia , Poliproteínas/genética , ARN Viral/sangre , Análisis de Secuencia de ARN
19.
Nat Commun ; 11(1): 6346, 2020 12 11.
Artículo en Inglés | MEDLINE | ID: mdl-33311465

RESUMEN

Acidothermus cellulolyticus CRISPR-Cas9 (AceCas9) is a thermophilic Type II-C enzyme that has potential genome editing applications in extreme environments. It cleaves DNA with a 5'-NNNCC-3' Protospacer Adjacent Motif (PAM) and is sensitive to its methylation status. To understand the molecular basis for the high specificity of AceCas9 for its PAM, we determined two crystal structures of AceCas9 lacking its HNH domain (AceCas9-ΔHNH) bound with a single guide RNA and DNA substrates, one with the correct and the other with an incorrect PAM. Three residues, Glu1044, Arg1088, Arg1091, form an intricate hydrogen bond network with the first cytosine and the two opposing guanine nucleotides to confer specificity. Methylation of the first but not the second cytosine base abolishes AceCas9 activity, consistent with the observed PAM recognition pattern. The high sensitivity of AceCas9 to the modified cytosine makes it a potential device for detecting epigenomic changes in genomes.


Asunto(s)
Actinobacteria/enzimología , Proteína 9 Asociada a CRISPR/química , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas , Edición Génica/métodos , Actinobacteria/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Cristalografía por Rayos X , Citosina , ADN/química , ADN/genética , ADN/metabolismo , Genoma , Secuenciación de Nucleótidos de Alto Rendimiento , Metilación , Modelos Moleculares , Conformación Proteica , ARN Guia/química
20.
Mol Biol (Mosk) ; 54(6): 968-974, 2020.
Artículo en Ruso | MEDLINE | ID: mdl-33276359

RESUMEN

The high variability of the influenza A virus poses a significant threat to public health, therefore monitoring viral strains and studying their genetic properties are important tasks. One part of this monitoring includes sequencing of influenza A viruses of any subtype and analysis of their whole genomes, which is especially important in cases of interspecies adaptation and reassortment. High-throughput sequencing technologies have significantly extended the capabilities of influenza virus epidemiological surveillance. The preparation stages for next generation sequencing (NGS) of influenza A virus include whole genome amplification using one-step RT-PCR, the results of which vary greatly depending on the sample type and quality, that, in turn, affects the coverage of virus fragments and the sequencing results in general. In this work, we propose to supplement the aforementioned technique of whole genome amplification of influenza A virus with sequential suppression PCRs to obtain an even coverage of viral segments of different lengths, which allows sequencing of samples with lower read coverage without decreasing the sequencing quality.


Asunto(s)
Genoma Viral , Secuenciación de Nucleótidos de Alto Rendimiento , Virus de la Influenza A , Virus de la Influenza A/genética , Reacción en Cadena de la Polimerasa
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