RESUMO
We characterize and extend a highly efficient method for constructing shotgun fragment libraries in which transposase catalyzes in vitro DNA fragmentation and adaptor incorporation simultaneously. We apply this method to sequencing a human genome and find that coverage biases are comparable to those of conventional protocols. We also extend its capabilities by developing protocols for sub-nanogram library construction, exome capture from 50 ng of input DNA, PCR-free and colony PCR library construction, and 96-plex sample indexing.
Assuntos
Fragmentação do DNA , Biblioteca Genômica , Reação em Cadeia da Polimerase/métodos , Análise de Sequência de DNA/métodos , Animais , DNA/isolamento & purificação , Drosophila/genética , Escherichia coli/genética , Éxons , Genoma Humano , Genoma de Inseto , Humanos , Transposases/metabolismoRESUMO
Rapid evolution and high intrahost sequence diversity are hallmarks of human and simian immunodeficiency virus (HIV/SIV) infection. Minor viral variants have important implications for drug resistance, receptor tropism, and immune evasion. Here, we used ultradeep pyrosequencing to sequence complete HIV/SIV genomes, detecting variants present at a frequency as low as 1%. This approach provides a more complete characterization of the viral population than is possible with conventional methods, revealing low-level drug resistance and detecting previously hidden changes in the viral population. While this work applies pyrosequencing to immunodeficiency viruses, this approach could be applied to virtually any viral pathogen.
Assuntos
Variação Genética , Genoma Viral , HIV/genética , Análise de Sequência de DNA/métodos , Vírus da Imunodeficiência Símia/genética , Sequência de Aminoácidos , Animais , HIV/química , HIV/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , Humanos , Macaca mulatta , Dados de Sequência Molecular , Alinhamento de Sequência , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/química , Vírus da Imunodeficiência Símia/imunologia , Especificidade da Espécie , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
We have cloned the chicken and mouse orthologues of the Caenorhabditis elegans heterochronic gene lin-41. During limb development, lin-41 is expressed in three phases over developmental time and most notably is associated with the developing autopod. Using chicken and mouse mutants and bead implantations, we report that lin-41 is genetically and biochemically downstream of both the Shh and Fgf signaling pathways. In C. elegans, it is proposed that lin-41 activity is temporally regulated by miRNAs (let-7 and lin-4) that bind to complementary sites in the lin-41 3'-untranslated region (UTR). Taking a bioinformatics approach, we also report the presence of potential miRNA binding sites in the 3'-UTR of chicken lin-41, including sites for the chicken orthologues of both C. elegans let-7 and lin-4. Finally, we show that these miRNAs and others are expressed in the chick limb consistent with the hypothesis that they regulate chicken Lin-41 activity in vivo.
Assuntos
Extremidades/embriologia , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Northern Blotting , Embrião de Galinha , Clonagem Molecular , Biologia Computacional , Componentes do Gene , Hibridização In Situ , Camundongos , Camundongos Mutantes , MicroRNAs/genética , Microesferas , Dados de Sequência Molecular , Alinhamento de Sequência , Análise de Sequência de DNA , Transdução de Sinais/genética , Especificidade da EspécieRESUMO
We have analyzed a new limb mutant in the chicken that we name oligozeugodactyly (ozd). The limbs of this mutant have a longitudinal postaxial defect, lacking the posterior element in the zeugopod (ulna/fibula) and all digits except digit 1 in the leg. Classical recombination experiments show that the limb mesoderm is the defective tissue layer in ozd limb buds. Molecular analysis revealed that the ozd limbs develop in the absence of Shh expression, while all other organs express Shh and develop normally. Neither Ptc1 nor Gli1 are detectable in mutant limb buds. However, Bmp2 and dHAND are expressed in the posterior wing and leg bud mesoderm, although at lower levels than in normal embryos. Activation of Hoxd11-13 occurs normally in ozd limbs but progressively declines with time. Phase III of expression is more affected than phase II, and expression is more severely affected in the more 5' genes. Interestingly, re-expression of Hoxd13 occurs at late stages in the distal mesoderm of ozd leg buds, correlating with formation of digit 1. Fgf8 and Fgf4 expression are initiated normally in the mutant AER but their expression is progressively downregulated in the anterior AER. Recombinant Shh protein or ZPA grafts restore normal pattern to ozd limbs; however, retinoic acid fails to induce Shh in ozd limb mesoderm. We conclude that Shh function is required for limb development distal to the elbow/knee joints, similar to the Shh(-/-) mouse. Accordingly we classify the limb skeletal elements as Shh dependent or independent, with the ulna/fibula and digits other than digit 1 in the leg being Shh dependent. Finally we propose that the ozd mutation is most likely a defect in a regulatory element that controls limb-specific expression of Shh.
