第二代RNA测序方法.ppt

RNA测序方法的进展 孟芳 2011.11.5 next-generation DNA sequencing (NGS) NGS领域的领头羊 Illumina, Roche 454, Helicos BioSciences and Life Technologies 四个公司 Several developments in RNA-seq methods improvements in transcription start site mapping strand-specific measurements gene fusion detection small RNA characterization detection of alternative splicing events a comparison of the different approaches that are available for each application and discuss the current limitations and the potential for future improvements Mapping transcription start sites (TSSs) 局限：the technology required high quantities of input RNA and generated only short reads (~20 nucleotides) per TSS. Gene fusion detection discussing two new developments in RNA-seq technologies: direct RNA sequencing (DRS)7 and methods for the reliable profiling of minute RNA quantities, which is important for translational research and clinical applications of RNA-seq. a | Single-molecule DNA and RNA sequencing technologies could be modified for single-cell applications. Cells can be delivered to flow cells using fluidics systems, followed by cell lysis and capture of mRNA species on the poly(dT)-coated sequencing surfaces by hybridization. Standard sequencing runs could take place on channels with a 127.5 mm2 surface area, requiring 2,750 images to be taken per cycle to image the entire channel area. The surface area needed to accommodate ~350,000 mRNA molecules contained in a single cell is ~0.4 mm2; thus, only eight images per cycle would be needed. Sequence analysis can be done with direct RNA sequencing (DRS)7or on-surface cDNA synthesis followed by single-molecule DNA sequencing26. b | Counter system workflow. Two probes are used for each target site: the capture probe (shown in red) contains a target-specific sequence and a modification that allows the immobilization of the molecules on a surface; the reporter probe contains a different target-specific sequence (shown in blue) and a fluorescent barcode (