Abstract: The third-generation sequencing technology has been widely used in genome research due to its advantage of long-fragment reads. The development of the sequencing technology was reviewed and the advantages and disadvantages of three different generations of sequencing technologies were summarized. Based on the third-generation sequencing, the methods of genome assembly including data pre-processing, sequence assembly, sequence repairing after assembly and chromosome localization methods were tracked and statistically analyzed. Application of sequencing technology and genome assembly in tobacco genome research was introduced.