The Whole Genome Sequencing And Analysis On The Strains Of Chinese Mycobacterium Tuberculosis EAI And CAS Genotypes

Objective To sequence the whole genome of a strain of Chinese Mycobacterium tuberculosis (MTB) EAI and CAS genotype isolates respectively and find out some specific characteristics of the genome of EAI and CAS genotype strains by comparing with the other MTB complex (MTBC) strains at the whole genome level, and offer data for the further research on virulence and molecular evolution of MTB.Methods Chinese MTB isolates CCDC06029 of EAI genotype and CCDC06153 of CAS genotype were selected from the clinical MTB isolates identified by drug susceptibility test (DST) and genotyping. The two isolates are sensitive to four first line drugs. Genomic DNA of the two clinical isolates were extracted by the CTAB method from the cultures of the two strains grown on Lowenstein-Jensen medium to their late log phases respectively. The complete genome sequencing was conducted with Roche 454 GS FLX system, and then the sequencing contigs were obtained by the GS de novo Assembler Software (http://www.454.com/). The relations of contigs were detemined by Blast 2.2.23.The regions of none sequence data were then filled in by sequencing the PCR products using the ABI 3730x1 capillary sequencers. Phred, Phrap and Consed software package were used for final edition and assembly, and low quality regions were resequenced. Then we used the whole genome sequence data to compared with other MTBC strain’s genome data which were acquired from the NCBI genome web site and try to find out the MTB EAI genotype and CAS genotype specific characteistics at the whole genome level.Results EAI genotype strain CCDC06029 and CAS genotype strain CCDC06153 were confirmed for the whole genome sequencing and analysis. The sizes of CCDC06029 and CCDC06153 are 4406422 bp and 4411683 bp, of which the sequencing accuracy is 99.9992% and 99.9995%, and the predicted protein coding sequences (CDSs) are 4118 and 4175, of which the average size of DNA sequence is 944bp and 941bp, respectively. The specific characters of EAI genotype and CAS genotype strains were obtained by comparing with other MTBC genotype strains which were downloaded from the NCBI genome website. By comparing with the sequence of H37Rv, we found 2967 SNPs and 430 Indels in EAI genotype strain CCDC06029, and 2213 SNPs and 374 Indels in CAS genotype strain CCDC06153. By comparing with the sequences of the other MTBC genotype strains, we found 1011 EAI specific SNPs and 723 CAS specific SNPs. There were 39 SNPs and 28 SNPs which might related to virulence genes in CCDC06029 and CCDC06153, respectively.Conclusion The whole genome sequencing on EAI genotype and CAS genotype strains have finished successfully, by which the whole genome sequence data have been provided to study the gene characteristics, pathogenic mechanism and molecular evolution of EAI and CAS genotype strains in future. The analysis results showed that the EAI and CAS genotype strains have some specific characters, such as SNPs; Indels, and so on, which will provide the bases to study on the origin and rapid identification of clinical isolates. It will promote us to recognize the biological hypostasis of MTBC, and has very importance for TB control and prevention.