微生物基因體分析平台-致病微生物Morganella morganii 基因體分析

Microbial genome analysis platform – Whole genome sequencing of Morganella morganii and identification of pathogenicity-related genes

陳昱廷(YU-TIN CHEN)

指導教授 ： 唐傳義

國立清華大學/電機資訊學院/資訊工程學系/博士(2012年)

https://doi.org/10.6843/NTHU.2012.00674

摘要

在次世代定序技術的發展，生命體遺傳解碼速度加快，解碼與基因相關分析工具也在累積，進行比較基因體分析，用於辨認物種內基因功能與可能致病原因。針對全序列定序之微生物基因體發展一套軟體分析平台，適合處理高通量且短序列的次世代定序。從原始序列產出，應用字串演算程式於品質、組裝、分析與找尋致病相關基因。在本地醫院患者分離排名第九於G(-)的伺機型微生物 Morganella morganii，有時會造成社區感染，菌血症，複雜型態泌尿道或膽管結石，且相關於患者插管。為了解它的基因體和致病機轉，從病患分離株KT，進行全序列測序且與相近物種進行比較基因體分析。基因體骨幹建置流程：嶄新組序(de novo)或貼序程式(re-sequence)配合組序混合策略，有許多字串演算法應用和發展於此。在案例， ABySS組序較優。組序KT菌後，搭配分析距離關係，得十二個連續序列。光學限制圖譜和分析顯示，最大的十個連續序列可以被並排。設計引子對、定序和補縫隙，來完成全基因體序列。案例A. baumannii CH 1-43，貼序配合組序混合策略，而剩下一個縫隙則可完成染色體全序列。基因體分析利用序列比對和累積的各物種序列為來源。包括基因預測，定義同源，功能分析和比較基因體分析差異流程：在Morganella morganii KT和Leptospira santarosai serovar Shermani，成功的揭露多種類致病基因，新基因結構和新基因的發現。Morganella morganii KT菌株共有3.8-Mb，其GC比例為51.15%，3565蛋白編碼基因，72個tRNA基因，和10個rRNA基因被預測和推定。致病相關基因含有抗藥因子，纖毛，鞭毛，鐵捕捉系統，第三型分泌系統等基因群，溶血基因和昆蟲毒素都是藉由比較14種相關的腸道科物種基因體得到。而乙醇胺利用基因組(eut operon)發現有19個基因組成，比原本腸道科17個基因更多，更有利於其潛藏於人體腸道內。M. morganii PC也進行全序列測序，約大KT 390-kb。分析指出有一個90-kb的ICE和ICEPm1相像，其它差異為數個prophage基因群。KT菌株是第一個被解開基因體密碼的M. morganii 物種並且有臨床致病力的菌株。比較基因體分析得到許多致病相關的基因和只在Proteeae亞科中發現的新基因。這基因體序列在細菌致病毒素與適存利基上提供了重要資訊。

關鍵字

基因體學 ； 微生物

並列摘要

As the sequencing technology advanced, genome of life decode also accelerate, related genome analysis tools also accumulated, comparative genomic analysis is current trend, which could applied to identify gene function and possible pathogenicity mechanism in organism. We develop a software pipeline for microbial genome project, which is suitable to high throughput and short read next generation sequencing analysis. The opportunistic enterobacterium, Morganella morganii, which can cause bacteraemia, is the ninth most prevalent cause of clinical infections in patients at local hospital. It is sometimes encountered in nosocomial settings and has been causally linked to catheter-associated bacteriuria, complex infections of the urinary and/or hepatobiliary tracts infection. To obtain insights into the genome biology of M. morganii and the mechanisms underlying its pathogenicity, the patient isolate strain KT was whole genome sequencing the genome and compared with the genome sequences of related bacteria. Genome backbone build rely on de novo assembly or re-sequence method, many string algorithm applications and developments. Genome backbone build: de novo assembler or re-sequencing compensate with assembler, a mix approach, many string algorithm applications and developments here. In cases, ABySS assembler performs better. Assembly results of two different distance libraries and scaffolder of M. morganii KT, twelve scaffolds remain. Align and fit ten scaffolds in the KT with optical map. PCR and sequencing for gap fill were conduct and toward the completion of the KT genome. In case, the A. baumannii CH 1-43 successful used mix approach and left one gap to complete genome. Genome analysis, use sequence alignment and accumulated organisms’ sequences as comparative genomics resources. The gene prediction, orthologous identification, functional analysis and comparative genomics analysis workflow: Morganella morganii KT and Leptospira santarosai serovar Shermani were successfully find pathogenicity or virulence related functional genes bring from those organisms and novel gene structures. The M. morganii KT possess genomic sequence of 3,826,919-bp sequence contained in 58 contigs has a GC content of 51.15% and includes 3,565 protein-coding sequences, 72 tRNA genes, and 10 rRNA genes. The pathogenicity-related genes encode determinants of drug resistance, fimbrial adhesins, an IgA protease, haemolysins, ureases, and insecticidal and apoptotic toxins as well as proteins found in flagellae, the iron acquisition system, a type-3 secretion system (T3SS), and several two-component systems. Comparison with 14 genome sequences from other members of Enterobacteriaceae revealed different degrees of similarity to several systems found in M. morganii. The most striking similarities were found in the IS4 family of transposases, insecticidal toxins, T3SS components, and proteins required for ethanolamine use (eut operon) and cobalamin (vitamin B12) biosynthesis. The eut operon and the gene cluster for cobalamin biosynthesis are not present in the other Proteeae genomes analysed. The second selected M. morganii PC strain were whole genome sequencing, and compare with KT, genome size are larger 390-kb in length. One ICE of 90-kb were similar ICE Pm1 were appeared, and several prophage gene clusters are mainly contribute to other differences. The strain KT is the first genome sequence of M. morganii, which is a clinically relevant pathogen. Comparative genome analysis revealed several pathogenicity-related genes and novel genes not found in the genomes of other members of Proteeae. Thus, the genome sequence of M. morganii provides important information concerning virulence and determinants of fitness in this pathogen. The M. morganii KT genome deposited in the DDBJ/EMBL/GenBank under the accession number ALJX00000000.

並列關鍵字

genomics ； microbial

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