靈芝(Ganoderma lucidum)是我國傳統中藥之一，近年來的研究也證實了靈芝中所含的多醣體、三萜類、以及其他微量元素具有抗癌、降血壓、治療肝病等許多的潛在療效。因此，靈芝全序列定序計畫有好的幫助對於靈芝二次代謝產物生合成的瞭解。

「次世代高通量DNA定序」方法開啟全基因體定序大門。靈芝全基因體計畫從2001年到至今，由台北榮民總醫院與國立陽明大學共同合作研究團隊，利用傳統的毛細管定序和新一代高通量定序技術所推動的。在靈芝全基因體序列長度在45MB左右內包含有13條染色體。我們利用基因預測工具Genemark-ES預測靈芝基因體含有14,025個預測基因，而這些基因我們利用暫定獨特基因(TUG)驗證基因預測工具預測出的基因結構。接著用NR及Uniprot序列資料庫作序列比對，註解出靈芝預測基因可能擁有的功能、存在的反應路徑。並且我去定義一些靈芝預測基因在KEGG反應路徑資料庫中，參與在萜類與固醇類生合成的反應路徑。

我們進一步討論靈芝全基因體組合的正確性，利用成對短序列(mate-pair short reads)及TUG可以提供基因體連續片段 (genomic contig)在每個基因體架構(genomic scaffold)內的順序。

Ling-Chi (Ganoderma lucidum), a chinese herb, The recent studies have revealed that polysaccharides, triterpenes, and some microelements in Ling-Chi, that has been reported to be effective in the treatment of neoplasia, hypertension, and hepatopathy. A whole genome sequence project for Ganoderma lucidum is well-studied to understand of biosynthesis of the secondary metabolites and some microelements.

Next-generation high-throughput DNA sequencing techniques are opening whole genome sequence assembled. Ling-Chi whole genome project sequenced using a combination of conventional capillary sequencer and new-generation sequencing technology, and was conducted by a collaboration of the team of the Veteran General Hospital-Taipei（VGH-Tapei）and the National Yang-Ming University（NYMU）from 2001 to present. The genome of Ling-Chi is about 45Mb in size and it contains 13 chromosomes. We predicted 14,052 genes by using gene prediction tool Genemark-ES in Ling-Chi. Moreover, these genes we used tentative unique genes (TUG) sequences applied to verify the gene structure predicted by prediction programs. The possible functions and reaction pathways of these predicted genes were annotated by using the sequence similarity to known proteins. And then, we identified some genes that might be involved in the terpenoid backbone and steroid biosynthesis by KEGG in Ling-Chi.

In additional to discussing the genome assembly correctness of Ling-Chi genome, using by mate-pair short reads and TUG sequences can be applied to provide the order of contigs in genomic scaffold.

誌謝 i
中文摘要 ii
ABSTRACT iii
CONTENTS iv
LIST OF FIGURES viii
LIST OF TABLES xi
Chapter 1 緒論 1
1.1 引言 1
1.2 靈芝 3
1.3 靈芝的生長週期 3
1.4 靈芝的外觀 4
1.4.1 孢子時期 4
1.4.2 菌絲體 4
1.4.3 靈芝子實體 5
1.5 靈芝多醣體 5
1.6 靈芝三萜類 6
1.7 靈芝基因體計畫 8
1.8 目前已有的真菌基因體計畫 10
1.8.1 Aspergillus niger全基因體計畫 10
1.8.2 Phytophthora infestans全基因體計畫 11
1.8.3 絲狀真菌醣基化反應途徑(glycosylation pathways)計畫 11
Chapter 2 第二章材料與方法 12
2.1 材料 12
2.1.1 硬體設備 12
2.1.1.1 計算資源： 12
2.1.1.2 儲存資源： 12
2.1.2 軟體配備 13
2.1.2.1 作業系統 13
2.1.2.2 軟體 13
2.2 資料來源 14
2.2.1 Uniprot Database 14
2.2.2 NR Database 14
2.2.3 靈芝表現序列(EST) 15
2.2.4 靈芝暫定獨特基因(TUG , Tentative Unique Gene) 15
2.2.5 靈芝基因體全序列 16
2.3 方法 17
2.3.1 分析平台−Linux 17
2.3.2 Perl 程式設計 17
2.3.3 BLAST 分析 18
2.3.3.1 資料庫格式化序列檔案 18
2.3.3.2 對序列資料庫作比對 19
2.3.4 大規模 BLAST 分析 20
2.3.5 Bioperl 模組進行資料格式轉換 20
2.3.6 SOAP 2.20 21
2.3.7 GeneMark-ES 22
2.3.8 Exonerate 23
2.3.9 KAAS ( KEGG Automatic Annotation Server ) 24
Chapter 3 第三章分析策略 25
3.1 靈芝全基因體組合序列品質評估 25
3.1.1 組合序列品質管理 25
3.2 基因預測 28
3.2.1 基因預測的品質管理 29
3.2.2 靈芝基因註解 31
Chapter 4 結果 33
4.1 靈芝基因數量 33
4.2 靈芝基因分析 35
4.2.1 高通量序列品質評估 35
4.2.2 全基因體序列品質評估 36
4.2.2.1 連續序列片段(contig)的品質評估 36
4.2.2.2 序列結構(scaffold) 39
4.2.3 基因預測品質評估 41
4.2.3.1 基因預測 41
4.2.3.2 預測基因品質評估 44
4.3 預測基因的註解分析 46
4.3.1 序列比對分析 46
4.3.2 多醣體生合成相關基因分析 47
4.3.3 三萜類生合成相關基因分析 50
4.3.4 重要基因可能參與多醣體及三萜類合成之相關基因 56
4.3.4.1 KRE6/SNK1 56
4.3.4.2 FKS1 60
4.3.4.3 HMG synthase 62
4.3.4.4 HMG reductase 64
4.3.4.5 FPP synthase 65
Chapter 5 第五章討論 68
5.1 靈芝全基因體 68
5.1.1 利用TUG可以幫助決定基因體scaffold內contig序列順序 68
5.2 靈芝基因預測 71
5.3 靈芝基因註解 71
參考文獻 72
附錄 77
利用 blast 對各個資料庫比對的結果 77
KRE6相關預測基因 78
FKS1相關預測基因 80
FPP synthase相關預測基因 81

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