中国结核分枝杆菌EAI和CAS基因型菌株全基因组测序分析

发布时间：2018-05-01 12:38

本文选题：结核分枝杆菌 + EAI基因型　；参考：《中国疾病预防控制中心》2012年硕士论文

【摘要】：目的对中国结核分枝杆菌EAI和CAS基因型临床分离菌株(CCDC06029和CCDC06153)全基因组进行测序,并与其它已经完成全基因组测序的结核分枝杆菌复合群菌株的数据进行比对分析,了解EAI和CAS基因型菌株的全基因组分子特征,为进一步深入研究,如微进化、毒力学等,提供基础。方法选取经细菌学、药物敏感性和分子分型鉴定的中国结核分枝杆菌EAI和CAS基因型临床分离菌株各1株,2株菌均对四种一线抗结核药物敏感。应用改良罗氏培养基培养,收集菌体,采用CTAB法提取全基因组DNA；用Roche 454 GS FLX测序仪进行全基因组高通量测序,测序结果使用DNA序列拼装软件进行初始序列拼装,得到测序重叠群(contigs),人工手动与参考序列进行blast比对后,确定各个测序重叠群位置关系和首尾连接方式并预测测序未覆盖区(gap区)的大小Gap区和测序低值区域,经特异性PCR扩增后,使用ABI 3730测序仪进行测序。基因组全长序列使用Phred、Phrap和Consed软件包进行拼装和碱基修正,最终得到CCDC06029和CCDC06153两株菌的全基因组序列数据,与其他从NCBI genome网站下载的结核分枝杆菌复合群菌株的全基因组数据,在全基因组水平进行比对、分析。结果测序得到中国结核分枝杆菌EAI和CAS基因型菌株CCDC06029(?)CCDC06153的全基因组完整序列,大小分别为4406422 bp和4411683 bp,其基因组测序数据的准确度分别为99.9992%和99.9995%,预测的蛋白质编码序列(predicted protein coding sequences, CDSs)分别为4118个和4175个,CDSs平均大小分别为944bp和941bp。通过与已完成全基因组测序的结核分枝杆菌复合群菌株的全基因组数据的比对分析发现了存在于中国结核分枝杆菌EAI和CAS基因型中的多种分子特征。EAI基因型测序菌株CCDC06029异于结核分枝杆菌H37Rv的SNP位点共有2967个,其中位于蛋白编码区的SNP有2650个；Indel数目为430个。CAS基因型测序菌株CCDC06153异于结核分枝杆菌H37Rv的SNP位点共有2213个,其中位于蛋白编码区的SNP有1802个；Indel数目为374个。在此次研究中发现1011个EAI基因型特异性SNP,其中852个SNP位于蛋白编码区内；发现723个CAS基因型特异性SNP,其中547个位于蛋白编码区内。EAI基因型菌株CCDC06029与结核分枝杆菌标准菌株H37Rv比对分析后,共发现39个可能与毒力基因相关的SNP位置,其中非同义突变有20个,同义突变有19个。CAS基因型菌株CCDC06153与结核分枝杆菌标准菌株H37Rv比对分析后,共发现28个可能与毒力基因相关的SNP位置,其中非同义突变有13个,同义突变有15个。结论成功完成了中国结核分枝杆菌EAI和CAS基因型菌株的全基因组测序,为进一步研究EAI和CAS基因型菌株的基因结构特征、致病机制、分子进化等提供了全基因组序列数据。全基因组数据的初步分析发现EAI和CAS基因型菌株基因组序列中存在某些特异性的Indel、SNP及其它分子生物学特征,将对EAI和CAS基因型的快速鉴定、溯源等多个方面的研究提供良好基础,并将促进对结核分枝杆菌生物学本质的认识,对于有效防治结核病具有重要意义。
[Abstract]:Objective to sequence the whole genome of Mycobacterium tuberculosis EAI and CAS genotype clinical isolates (CCDC06029 and CCDC06153), and to compare and analyze the data of other Mycobacterium tuberculosis complex strains which have completed the whole genome sequencing, and to understand the whole genomic characteristics of the EAI and CAS genotypes, so as to further deepen the study of the genome of the strains of EAI and CAS. Studies, such as microevolution, toxic mechanics, etc., provide the basis. Methods 1 strains of Mycobacterium tuberculosis EAI and CAS genotypes were selected by bacteriology, drug sensitivity and molecular typing, and 2 strains were sensitive to four kinds of first-line anti tuberculosis drugs. The improved Roche culture medium was used to collect the mycelium and the whole base was extracted by CTAB method. The whole genome high throughput sequencing was carried out with the Roche 454 GS FLX sequencer. The sequencing results were sequenced using the DNA sequence assembly software for the initial sequence assembly, and the sequencing overlap group (contigs) was obtained. After the manual manual and the reference sequence was compared to the sequence, the location relationship and the first and the tail connection mode of the sequencing overlaps were determined and the sequencing was predicted. The size Gap area of the cover area (gap area) and the sequence of low value regions were amplified by specific PCR and were sequenced using ABI 3730 sequencer. The whole genome sequence used Phred, Phrap and Consed software packages to assemble and modify the base. Finally, the whole genome sequence data of the two strains of CCDC06029 and CCDC06153 were obtained, and the other from the NCBI genome web site. The whole genome data of the Mycobacterium tuberculosis complex strains were compared and analyzed at the whole genome level. The whole genome sequence of EAI and CAS genotype CCDC06029 (?) CCDC06153 of Mycobacterium tuberculosis in China was sequenced, the size of the genome was 4406422 BP and 4411683 BP respectively. The accuracy of the genome sequencing data was respectively For 99.9992% and 99.9995%, the predicted protein coding sequences (predicted protein coding sequences, CDSs) were 4118 and 4175 respectively. The average CDSs size of 944bp and 941bp., respectively, was found in Chinese tuberculosis through comparison and analysis of all genome data of the genome of Mycobacterium tuberculosis with complete genome sequencing. A variety of molecular characteristics in the EAI and CAS genotypes of Mycobacterium tuberculosis,.EAI genotypes, CCDC06029 are different from SNP loci of Mycobacterium tuberculosis H37Rv, of which there are 2650 SNP in the protein coding region; Indel number is 430.CAS genotype sequencing strains, CCDC06153 is different from the H37Rv SNP loci of Mycobacterium tuberculosis. There were 1802 SNP in the protein coding region and 374 in the Indel number. In this study, 1011 EAI genotype specific SNP were found, of which 852 SNP were located in the protein coding region; 723 CAS genotype specific SNP were found, and 547 of the.EAI genotype strains were located in the protein coding region, CCDC06029 and the standard Mycobacterium tuberculosis After H37Rv comparison, 39 SNP locations associated with virulence genes were found, of which there were 20 non synonymous mutations, and 19.CAS genotype strains, CCDC06153, and standard strains of Mycobacterium tuberculosis were compared and analyzed by the synonymous mutation, and a total of 28 possible locations of virulence genes were found, including 13 non synonymous mutations, and synonyms. There are 15 mutations. Conclusion the whole genome sequence of EAI and CAS genotypes of Mycobacterium tuberculosis in China has been successfully completed. The whole genome sequence data are provided for further research on the gene structure characteristics of EAI and CAS genotype strains, the pathogenesis mechanism and the molecular evolution. The preliminary analysis of the whole genome data has found the EAI and CAS genotypes. The existence of certain specific Indel, SNP and other molecular biological characteristics in the sequence of the group will provide a good basis for the rapid identification of EAI and CAS genotypes, traceability and so on, and will promote the understanding of the biological nature of Mycobacterium tuberculosis, which is of great significance for the effective prevention and treatment of tuberculosis.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378.911

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