ObjectiveTo conduct the sequencing and preliminarily analysis of the whole genome of BCG Shanghai D₂PB302 strain （hereinafter referred to as BCG Shanghai D₂ strain）， which has been used exclusively for the vaccine production in China. MethodsThe DNA of of BCG Shanghai D₂ strain （D2-JIA12-1） was extracted， and the whole genome was sequenced by Pacbio-RS Ⅱ. The sequence data was assembled by Smrtlink and polished with the illumina data. Genes， tRNA and rRNA were predicted based on the sequence data. The functional annotation of predicted genes was performed through BLASTP. The IVE-TB antigen gene and MTBVAC were selected as the target sequences to be compared with Mycobacterium tuberculosis H37Rv （NC_000962.3）. ResultsThe sequence length of BCG Shanghai D₂ strain was 4 045 232 bp， and the GC content was 65.66%. A total of 4 259 protein-encoding genes were predicted， with an average gene size of 933 bp. 2 476 genes had biological functions and others were hypothetical proteins.144 virulence genes were obtained by comparing with the VFDB. There were 29 type Ⅶ secretion system genes and 10 PE/PPE protein family genes. ConclusionThe whole genome sequence of BCG Shanghai D₂ strain is clarified. It lays a broad foundation for subsequent detection of the stability of major antigen genes.