We resequenced tobacco genome for a corporate client. The gDNA was isolated, and TruSeq DNA library of different insert sizes was prepared. We sequenced these libraries on HiSeq platform. After preprocessing of the data, genome alignment was performed. We called the SNPs using variant calling pipeline developed by team Nucleome, especially for this project. The discovered SNPs were filtered based on different parameters, and high-quality SNPs were used for development of Custom SNP chip and genotyping on GeneTitan system.