Abstract: Pyrosequencing is a real-time DNA sequencing technology based on pyrophosphate (PPi) detection without the need of electrophoresis and fluorescence labeling.It is characterized by high-quantitative performance,accurate analysis and easy automation.The bottlenecks in its application include:low sensitivity and high cost of reagents,expensive instrumentation,and tedious process in the preparation of sequencing template.Recently,we developed a set of methods to solve the bottlenecks in pyrosequencing,including highly sensitive sequencing reagents,a portable instrument,and a simple way for preparing sequencing template.These methods have been successfully applied in molecular diagnosis,such as the sequence verification of bacteria,virus and genetically modified transgenic organisms,the detection of single nucleotide polymorphisms,copy number variation,gene mutations,and the quantification of microRNA and mRNA,and in pharmacogenomic studies.The results indicated that the platform has the advantages of high sensitivity,small size of instrument,low cost and simple operation.This paper describes our main contributions in pyrosequencing technology and novel applications of the pyrosequencing platforms in genetic analysis.