The Whole Genome Sequencing And Preliminary Analysis Of Two Mycobacterium Tuberculosis Beijing Genotype Strains

Objective To finish the whole genome sequencing of two typical Mycobacterium tuberculosis (MTB) Beijing family (Beijing genotype) isolates (CCDC5079and CCDC5180) and try to find out some specific characteristics of the Beijing genotype strains by comparing with the other MTB complex (MTBC) strains at the whole genome level, and try to find some anti-TB drug resistance-associated genomic markers, offer data for the further research on MTB Beijing family.Methods Two typical clinical MTB Beijing genotype isolates were selected from the clinical MTB isolates identified by drug susceptibility test (DST) and genotyping, including apiece one of sensitivity and resistance to three first line drugs (Isoniazid, Rifarmpicin and Streptomycin). Genomic DNA of the two clinical strains were extracted by the CTAB method from these strains grown on Lowenstein-Jensen medium to their late log phases.The complete genome sequencing was conducted with Roche454GS FLX system, and then the sequencing contigs were obtained employing the GS de novo Assembler Software (http://www.454.com/). The regions of none sequence data were then filled in by sequencing the PCR products using by means of the ABI3730x1capillary sequencers. Phred, Phrap and Consed software package were used for final assembly and edition, and low quality region were resequenced. Then use the whole genome sequence data compared with other MTBC strain’s genome data which were acquired from the NCBI genome web site and try to find out the MTB Beijing genotype specific characteistics or other differences at the whole genome level.Results Two typecal Beijing genotype strains CCDC5079and CCDC5180, which is sensitivity and resistance to the three first line drugs respectively, were confirmed for the whole genome sequencing and analysis. The whole genome sequence of the two typecal Beijing genotype strains were obtained, and the sequencing accuracy of the2genome of CCDC5079and CCDC5180is99.9996%and99.9988%, and the predicted protein coding sequences (CDSs) are4158and4161, of which the average of DNA sequence is971bp and970bp, respectively. The Beijing genotype specific characters were obtained by compareing with other8MTBC strains which were downloaded from the NCBI genome website, the Beijng family specific moleculer markers contains609Beijing genotype specific SNPs (single nuclear acid polymorphsims),84Idels (insertion and deletions),156SNPs might asssociate to some anti-TB drug resistance of MTBC, and other characteristics of MTB Beijing genotype.Conclusion The whole genome sequencing on the two typecal Beijing genotype strains have finished successfully, by which the whole genome sequence data have been provided to study the gene characteristics and molecular evolution of MTB Beijing family in future. The preliminary analysis results showed that the MTB Beijing genotype strains have some specific characters, such as SNPs, Indels, and so on, by which the bases has been provided to study on the rapid identification of clinical isolates, drug sensitive test and the mechanism of drug resistance and pathogenesis. It is significant importance for achieving the target of TB control and prevention.