RESUMO
BACKGROUND: Next generation sequencing (NGS) technologies have improved the study of hereditary diseases. Since the evaluation of bioinformatics pipelines is not straightforward, NGS demands effective strategies to analyze data that is of paramount relevance for decision making under a clinical scenario. According to the benchmarking framework of the Global Alliance for Genomics and Health (GA4GH), we implemented a new simple and user-friendly set-theory based method to assess variant callers using a gold standard variant set and high confidence regions. As model, we used TruSight Cardio kit sequencing data of the reference genome NA12878. This targeted sequencing kit is used to identify variants in key genes related to Inherited Cardiac Conditions (ICCs), a group of cardiovascular diseases with high rates of morbidity and mortality. RESULTS: We implemented and compared three variant calling pipelines (Isaac, Freebayes, and VarScan). Performance metrics using our set-theory approach showed high-resolution pipelines and revealed: (1) a perfect recall of 1.000 for all three pipelines, (2) very high precision values, i.e. 0.987 for Freebayes, 0.928 for VarScan, and 1.000 for Isaac, when compared with the reference material, and (3) a ROC curve analysis with AUC > 0.94 for all cases. Moreover, significant differences were obtained between the three pipelines. In general, results indicate that the three pipelines were able to recognize the expected variants in the gold standard data set. CONCLUSIONS: Our set-theory approach to calculate metrics was able to identify the expected ICCs related variants by the three selected pipelines, but results were completely dependent on the algorithms. We emphasize the importance to assess pipelines using gold standard materials to achieve the most reliable results for clinical application.
Assuntos
Biologia Computacional , Sequenciamento de Nucleotídeos em Larga Escala , Benchmarking , Biologia Computacional/métodos , Biologia Computacional/normas , Bases de Dados Genéticas , Predisposição Genética para Doença/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , SoftwareRESUMO
RNA sequencing (RNA-seq) coupled to DNA methylation strategies enables the detection and characterization of genes which expression levels might be mediated by DNA methylation. Here we describe a bioinformatics protocol to analyze gene expression levels using RNA-seq data that allow us to identify candidate genes to be tested by bisulfite assays. The candidate methylated genes are usually those that are low expressed in a particular condition or developmental stage.
Assuntos
Metilação de DNA , Grão Comestível/genética , Perfilação da Expressão Gênica , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Transcriptoma , Biologia Computacional/métodos , Bases de Dados Genéticas , Perfilação da Expressão Gênica/métodos , Perfilação da Expressão Gênica/normas , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Análise de Sequência de DNA/métodos , Software , Zea mays/genéticaRESUMO
This study compared imputation from lower-density commercial and customized panels to high-density panels and a combined panel (Illumina and Affymetrix) in Nelore beef cattle. Additionally, linkage disequilibrium and haplotype block conformation were estimated in individual high-density panels and compared with corresponding values in the combined panel after imputation. Overall, 814 animals were genotyped using BovineHD BeadChip (IllumHD), and 93 of these animals were also genotyped using the Axion Genome-Wide BOS 1 Array Plate (AffyHD). In general, customization considering linkage disequilibrium and minor allele frequency had the highest accuracies. The IllumHD panel had higher values of linkage disequilibrium for short distances between SNPs than AffyHD and the combined panel. The combined panel had an increased number of small haplotype blocks. The use of a combined panel is recommended due to its increased density and number of haplotype blocks, which in turn increase the probability of a marker being close to a quantitative trait locus of interest. Considering common SNPs between IllumHD and AffyHD for the customization of a low-density panel increases the imputation accuracy for IllumHD, AffyHD and the combined panel.
Assuntos
Bovinos/genética , Estudo de Associação Genômica Ampla/métodos , Técnicas de Genotipagem/métodos , Animais , Frequência do Gene , Estudo de Associação Genômica Ampla/normas , Técnicas de Genotipagem/normas , Sequenciamento de Nucleotídeos em Larga Escala/normas , Desequilíbrio de Ligação , Polimorfismo de Nucleotídeo Único , Locos de Características QuantitativasRESUMO
AIM: The aim of this research was to create a work scheme for the isolation of the different micro-organisms commonly found in hydrogen-producing reactors and to test its effectiveness. METHODS AND RESULTS: Methods were selected to isolate anaerobic spore-forming fermenters, anaerobic fermenters that do not form spores, facultative aerobic fermenters and lactic acid bacteria. The methods were tested in two samples taken from a hydrogen-producing reactor fed with cheese whey. 16S rRNA gene sequences from isolates were compared with pyrosequencing analysis from the same samples. The isolates represented more than 88% of the abundance detected by pyrosequencing. Organisms from the genera Clostridium, Rahnella, Megasphaera, Lactobacillus, Propionibacterium, Bifidobacterium, Chryseobacterium and Acetobacter were isolated. Hydrogen-producing capacity was confirmed for the Clostridium, Rahnella and Megasphaera isolates. Coculture experiments indicate that Megasphaera prevented the total inhibition of Clostridium by Lactobacillus. CONCLUSION: The work scheme proposed was effective to isolate most of the micro-organisms detected by pyrosequencing analysis. Physiological studies suggested a key role of Megasphaera. SIGNIFICANCE AND IMPACT OF THE STUDY: We showed the high culturability of the microbial communities from hydrogen-producing bioreactors. The isolates can be used to perform physiological studies to understand the H2 -producing process.
Assuntos
Bactérias/isolamento & purificação , Reatores Biológicos/microbiologia , Hidrogênio , Fluxo de Trabalho , Bactérias/classificação , Bactérias/genética , Queijo/microbiologia , Técnicas de Cocultura , DNA Bacteriano/genética , Sequenciamento de Nucleotídeos em Larga Escala/normas , Interações Microbianas , RNA Ribossômico 16S/genética , Soro do Leite/microbiologiaRESUMO
Genome assembly depends critically on read length. Two recent technologies, from Pacific Biosciences (PacBio) and Oxford Nanopore, produce read lengths >20 kb, which yield de novo genome assemblies with vastly greater contiguity than those based on Sanger, Illumina, or other technologies. However, the very high error rates of these two new technologies (â¼15% per base) makes assembly imprecise at repeats longer than the read length and computationally expensive. Here we show that the contiguity and quality of the assembly of these noisy long reads can be significantly improved at a minimal cost, by leveraging on the low error rate and low cost of Illumina short reads. Namely, k-mers from the PacBio raw reads that are not present in Illumina reads (which account for â¼95% of the distinct k-mers) are deemed sequencing errors and ignored at the seed alignment step. By focusing on the â¼5% of k-mers that are error free, read overlap sensitivity is dramatically increased. Of equal importance, the validation procedure can be extended to exclude repetitive k-mers, which prevents read miscorrection at repeats and further improves the resulting assemblies. We tested the k-mer validation procedure using one long-read technology (PacBio) and one assembler (MHAP/Celera Assembler), but it is very likely to yield analogous improvements with alternative long-read technologies and assemblers, such as Oxford Nanopore and BLASR/DALIGNER/Falcon, respectively.
Assuntos
Mapeamento de Sequências Contíguas/métodos , Mapeamento de Sequências Contíguas/normas , Algoritmos , Animais , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Nanoporos , Análise de Sequência de DNA/métodos , Análise de Sequência de DNA/normasRESUMO
The aim of this study was to develop microsatellite markers as a tool to study population structure, genetic diversity and effective population size of Echinopsis chiloensis, an endemic cactus from arid and semiarid regions of Central Chile. We developed 12 polymorphic microsatellite markers for E. chiloensis using next-generation sequencing and tested them in 60 individuals from six sites, covering all the latitudinal range of this species. The number of alleles per locus ranged from 3 to 8, while the observed (Ho) and expected (He) heterozygosity ranged from 0.0 to 0.80 and from 0.10 to 0.76, respectively. We also detected significant differences between sites, with FST values ranging from 0.05 to 0.29. Microsatellite markers will enable us to estimate genetic diversity and population structure of E. chiloensis in future ecological and phylogeographic studies.
Assuntos
Cactaceae/genética , Sequenciamento de Nucleotídeos em Larga Escala/normas , Análise de Sequência de DNA/normas , DNA de Plantas/genética , Genes de Plantas , Repetições de Microssatélites , Filogenia , Padrões de ReferênciaRESUMO
The application of next-generation sequencing (also known as deep sequencing or massively parallel sequencing) for adventitious agent detection is an evolving field that is steadily gaining acceptance in the biopharmaceutical industry. In order for this technology to be successfully applied, a robust method that can isolate viral nucleic acids from a variety of biological samples (such as host cell substrates, cell-free culture fluids, viral vaccine harvests, and animal-derived raw materials) must be established by demonstrating recovery of model virus spikes. In this report, we implement the sample preparation workflow developed by Feng et. al. and assess the sensitivity of virus detection in a next-generation sequencing readout using the Illumina MiSeq platform. We describe a theoretical model to estimate the detection of a target virus in a cell lysate or viral vaccine harvest sample. We show that nuclease treatment can be used for samples that contain a high background of non-relevant nucleic acids (e.g., host cell DNA) in order to effectively increase the sensitivity of sequencing target viruses and reduce the complexity of data analysis. Finally, we demonstrate that at defined spike levels, nucleic acids from a panel of model viruses spiked into representative cell lysate and viral vaccine harvest samples can be confidently recovered by next-generation sequencing.