RESUMEN
Despite remarkable progress in DNA sequencing technologies there remains a trade-off between short-read platforms, having limited ability to sequence homopolymers, repeated motifs or long-range structural variation, and long-read platforms, which tend to have lower accuracy and/or throughput. Moreover, current methods do not allow direct readout of epigenetic modifications from a single read. With the aim of addressing these limitations, we have developed an optical electrowetting sequencing platform that uses step-wise nucleotide triphosphate (dNTP) release, capture and detection in microdroplets from single DNA molecules. Each microdroplet serves as a reaction vessel that identifies an individual dNTP based on a robust fluorescence signal, with the detection chemistry extended to enable detection of 5-methylcytosine. Our platform uses small reagent volumes and inexpensive equipment, paving the way to cost-effective single-molecule DNA sequencing, capable of handling widely varying GC-bias, and demonstrating direct detection of epigenetic modifications.
Asunto(s)
ADN/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Análisis de Secuencia de ADN/métodos , Imagen Individual de Molécula , Composición de Base/genética , Humanos , Nanotecnología , Nucleótidos/genéticaRESUMEN
Varicella zoster virus (VZV) becomes latent in ganglionic neurons derived from neural crest cells. Because the adrenal gland also contains medullary chromaffin cells of neural crest origin, we examined human adrenal glands and medullary chromaffin cell tumors (pheochromocytomas) for VZV and herpes simplex virus type 1 (HSV-1). We found VZV, but not HSV-1, DNA in 4/63 (6 %) normal adrenal glands. No VZV transcripts or antigens were detected in the 4 VZV DNA-positive samples. No VZV or HSV-1 DNA was found in 21 pheochromocytomas.