RESUMO
Metabarcoding DNA sequencing has revolutionized the study of microbial communities. Third-generation sequencing producing long reads had opened up new perspectives. Obtaining the full-length ribosomal RNA gene would permit one to reach a better taxonomic resolution at the species or the strain level. However, Oxford Nanopore Technologies (ONT) sequencing produces reads with high error rates, which introduces biases in analysis. Understanding the biases introduced during the analysis allows one to better interpret the biological results and take care of conclusions drawn from metabarcoding experiments. To benchmark an analysis process, the ground truth, i.e., the real composition of the microbial community, has to be known. In addition to artificial mock communities, simulated data are often used to evaluate the biases and performances of the bioinformatics analysis step. Currently, no specific tool has been developed to simulate metabarcoding long reads, mimic the error rate and the length distribution, and allow one to benchmark the analysis process. Here, we introduce CuReSim-LoRM, for the customized read simulator to generate long reads for metabarcoding. We showed that CuReSim-LoRM is able to produce reads with varying error rates and length distributions by mimicking the real data very well.
Assuntos
Microbiota , Nanoporos , Benchmarking , Biologia Computacional , Análise de Sequência de DNARESUMO
Cellular stress can influence efficiency of iPSCs generation and their differentiation. However, the role of intracellular cytoprotective factors in these processes is still not well known. Therefore, we investigated the effect of HO-1 (Hmox1) or Nrf2 (Nfe2l2), two major cytoprotective genes. Hmox1-/- fibroblasts demonstrated decreased reprogramming efficiency in comparison to Hmox1+/+ cells. Reversely, pharmacological enhancement of HO-1 resulted in higher number of iPSCs colonies. Importantly, elevated level of both p53 and p53-regulated miR-34a and 14-3-3σ was observed in HO-1-deficient fibroblasts whereas downregulation of p53 in these cells markedly increased their reprogramming efficiency. In human fibroblasts HO-1 silencing also induced p53 expression and affected reprogramming outcome. Hmox1+/+ and Hmox1-/- iPSCs similarly differentiated in vitro to cells originating from three germ layers, however, lower number of contracting cells was observed during this process in HO-1-deficient cells indicating attenuated cardiac differentiation. Importantly, silencing of Hmox1 in murine ESC using CRISPR/Cas-9 editing also impaired their spontaneous cardiac differentiation. Decreased reprogramming efficiency was also observed in Nrf2-lacking fibroblasts. Reversely, sulforaphane, a Nrf2 activator, increased the number of iPSCs colonies. However, both Nfe2l2+/+ and Nfe2l2-/- iPSCs showed similar pluripotency and differentiation capacity. These results indicate that regulation of HO-1 expression can further optimize generation and cardiac differentiation of iPSCs. © 2018 IUBMB Life, 70(2):129-142, 2018.
Assuntos
Diferenciação Celular/fisiologia , Técnicas de Reprogramação Celular/métodos , Heme Oxigenase-1/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Proteínas de Membrana/metabolismo , Animais , Ciclo Celular/fisiologia , Fibroblastos , Heme Oxigenase-1/genética , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/citologia , Miócitos Cardíacos/fisiologia , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
HLA-B*35:29:03 differs from HLA-B*35:29:01 by one nucleotide substitution at position 374 in exon 2.