RESUMO
Sequence capture methods for targeted next generation sequencing promise to massively reduce cost of genomics projects compared to untargeted sequencing. However, evaluated capture methods specifically dedicated to biologically relevant genomic regions are rare. Whole exome capture has been shown to be a powerful tool to discover the genetic origin of disease and provides a reduction in target size and thus calculative sequencing capacity of >90-fold compared to untargeted whole genome sequencing. For further cost reduction, a valuable complementing approach is the analysis of smaller, relevant gene subsets but involving large cohorts of samples. However, effective adjustment of target sizes and sample numbers is hampered by the limited scalability of enrichment systems. We report a highly scalable and automated method to capture a 480 Kb exome subset of 115 cancer-related genes using microfluidic DNA arrays. The arrays are adaptable from 125 Kb to 1 Mb target size and/or one to eight samples without barcoding strategies, representing a further 26 - 270-fold reduction of calculative sequencing capacity compared to whole exome sequencing. Illumina GAII analysis of a HapMap genome enriched for this exome subset revealed a completeness of >96%. Uniformity was such that >68% of exons had at least half the median depth of coverage. An analysis of reference SNPs revealed a sensitivity of up to 93% and a specificity of 98.2% or higher.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Sequência de DNA/métodos , Éxons , Genômica/métodos , Humanos , Polimorfismo de Nucleotídeo Único , Alinhamento de Sequência/métodosRESUMO
Failure of pathogenic fungi to breach the plant cell wall constitutes a major component of immunity of non-host plant species--species outside the pathogen host range--and accounts for a proportion of aborted infection attempts on 'susceptible' host plants (basal resistance). Neither form of penetration resistance is understood at the molecular level. We developed a screen for penetration (pen) mutants of Arabidopsis, which are disabled in non-host penetration resistance against barley powdery mildew, Blumeria graminis f. sp. hordei, and we isolated the PEN1 gene. We also isolated barley ROR2 (ref. 2), which is required for basal penetration resistance against B. g. hordei. The genes encode functionally homologous syntaxins, demonstrating a mechanistic link between non-host resistance and basal penetration resistance in monocotyledons and dicotyledons. We show that resistance in barley requires a SNAP-25 (synaptosome-associated protein, molecular mass 25 kDa) homologue capable of forming a binary SNAP receptor (SNARE) complex with ROR2. Genetic control of vesicle behaviour at penetration sites, and plasma membrane location of PEN1/ROR2, is consistent with a proposed involvement of SNARE-complex-mediated exocytosis and/or homotypic vesicle fusion events in resistance. Functions associated with SNARE-dependent penetration resistance are dispensable for immunity mediated by race-specific resistance (R) genes, highlighting fundamental differences between these two resistance forms.
Assuntos
Proteínas de Arabidopsis/imunologia , Arabidopsis/imunologia , Parede Celular/imunologia , Fungos/imunologia , Hordeum/imunologia , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Proteínas de Transporte Vesicular , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Parede Celular/microbiologia , Clonagem Molecular , Hordeum/citologia , Hordeum/genética , Hordeum/microbiologia , Proteínas de Membrana/química , Proteínas de Membrana/genética , Dados de Sequência Molecular , Mutação , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/imunologia , Proteínas do Tecido Nervoso/metabolismo , Doenças das Plantas/microbiologia , Ligação Proteica , Proteínas Qa-SNARE , Proteínas SNARE , Proteína 25 Associada a Sinaptossoma , Técnicas do Sistema de Duplo-HíbridoRESUMO
We report a flexible method for selective capture of sequence fragments from complex, eukaryotic genome libraries for next-generation sequencing based on hybridization to DNA microarrays. Using microfluidic array architecture and integrated hardware, the process is amenable to complete automation and does not introduce amplification steps into the standard library preparation workflow, thereby avoiding bias of sequence distribution and fragment lengths. We captured a discontiguous human genomic target region of 185 kb using a tiling design with 50mer probes. Analysis by high-throughput sequencing using an Illumina/Solexa 1G Genome Analyzer revealed 2150-fold enrichment with mean per base coverage between 4.6 and 107.5-fold for the individual target regions. This method represents a flexible and cost-effective approach for large-scale resequencing of complex genomes.