RESUMO
The recent development of a semiconductor-based, non-optical DNA sequencing technology promises scalable, low-cost and rapid sequence data production. The technology has previously been applied mainly to genomic sequencing and targeted re-sequencing. Here we demonstrate the utility of Ion Torrent semiconductor-based sequencing for sensitive, efficient and rapid chromatin immunoprecipitation followed by sequencing (ChIP-seq) through the application of sample preparation methods that are optimized for ChIP-seq on the Ion Torrent platform. We leverage this method for epigenetic profiling of tumour tissues.
Assuntos
Genoma Humano , Histonas/metabolismo , Melanoma/química , Processamento de Proteína Pós-Traducional , Análise de Sequência de DNA/instrumentação , Neoplasias Cutâneas/química , Animais , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Epigênese Genética , Feminino , Histonas/genética , Humanos , Melanoma/genética , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Semicondutores , Análise de Sequência de DNA/métodos , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismoRESUMO
We present an automated, high throughput library construction process for 454 technology. Sample handling errors and cross-contamination are minimized via end-to-end barcoding of plasticware, along with molecular DNA barcoding of constructs. Automation-friendly magnetic bead-based size selection and cleanup steps have been devised, eliminating major bottlenecks and significant sources of error. Using this methodology, one technician can create 96 sequence-ready 454 libraries in 2 days, a dramatic improvement over the standard method.
Assuntos
Processamento Eletrônico de Dados , Biblioteca Gênica , Ensaios de Triagem em Larga Escala , Análise de Sequência de DNA/métodos , Algoritmos , Humanos , MicroesferasRESUMO
This study systematically examined the viral long control region (LCR) activities and their responses to E2 for human papillomavirus (HPV) types 11, 16, and 18 as well as bovine papillomavirus 1 (BPV1) in a number of different cell types, including human cervical cancer cell lines, human oral keratinocytes, BJ fibroblasts, as well as CV1 cells. The study revealed cell- and virus-type specific differences among the individual LCRs and their regulation by E2. In addition, the integration of the LCR into the host genome was identified as a critical determinant for LCR activity and its response to E2. Collectively, these data indicate a more complex level of transcriptional regulation of the LCR by cellular and viral factors than previously appreciated, including a comparatively low LCR activity and poor E2 responsiveness for HPV16 in most human cells. This study should provide a valuable framework for future transcriptional studies in the papillomavirus field.
