| This study consists of two sections. And the first one is review of sequencing the whole genome of Bt YBT1520. The protocol determined for this project is WGS—whole genome shotgun. The first step of WGS is after constructing the PUC18 and pBeloBAC11 DNA libraries, sequence the two ends with universal primers SP6 and T7. As a large amount of reads are acquired, the program phred transformed the trace file of reads to digital file with the extention name "phd". Later, program phd2fasta changed them into fasta format with two kinds of files--one contained sequence information, and the other for quality respectively. The program phrap assemblied those reads into a serial of contigs and the team of sequencer detected the relation between contigs, by the constrainal length and orientation of reads. So as to design the target primers and sequence the gap, this process is called Finishing or gap-filling. It never ends until, the chronomosome and all plasmids are sequenced circlely. There are four phases for this job: 1) the number of gap droped from 2000 to 1400. Major method is sequencing another more PUC18 libraries. 2) 1400-400, Major method is filling the gaps according the orientation relation of PUC reads. 3) 400-100, filling the gaps according to the BAC relation and reference genome. 4) 100—8 at present, filling the gap according to the fosmid library, reference genome and random PCR and sequencing with PCR design of ends of contigs. From the experience and data character of this case, several results are found as follow. 1) The coverage depth of multi-copy plasmids are ten time than chromosome. 2) A lot of repeats cases arose, because there are a lot cases of multi-copy or segment of genes are located on the this same genome,such as rDNA, Cry, IS, Tn family so on. Key point is the copy or segment lengths are far longer than 500bp-700bp the maximal range of sequencing capacity. In case of those fragment, phrap can't put reads on the right sites of contigs. 3) The PUC18 and production of PCR are good as the templates for sequcencing, the pBeloBAC11 is OK. But the total genome background is not good.The second section of this study is about evolution and taxonomy of Bc group that Bt YBT1520 belongs to. And a taxon probe—16S rDNA was analysised. Firstly review the MEE MISE AFLE alplication on this group. For the 16S rDNA job, research focus on strains which has genome data on NCBI. Until now, 8 whole genomes in this group have been accomplished and the data can been acquired from NCBI. These 8 whole genomes data were the main source for this paper. And 16S rDNA is a "gold standard" in the' field of bacterial identification. Their 98 16S rDNA in this 8 genomes are under research, and the global minimal identity within the genome is 99.27%, except one sequence in Bacillus cereus ATCC 10987, because it is pretty special and around 50bp longer than others. The digital 99.72% is the local minimal identity by blastn program and the respective match length is 1417 bp, which means they share almost the same functional 16S rDNA and should be one species. For the taxa aspect, Bacillus subtilis is closest to the Bc group. Considering the RNA operon, the number of Bc group ranks first on the bacterial cases, which reflects the the potential ecological ability. As for RNA operons sitation on the chromosome, One RNA operon locates on the negative strand near to the ori, and the rest are the positive strand near to the ori. The former RNA with rich tRNA gene behind and RNAP around, which means this region function the translation mainly.
|
PDF Full Text Request