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Application of next-generation sequencing technologies in functional genomics. 生 科 102 甲 498062076 吳亞璇. Advances in DNA sequencing technologies. Sanger sequencing 454 sequencing technology Illumina ABI/ SOLiD. Sanger sequencing.

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application of next generation sequencing technologies in functional genomics

Application of next-generation sequencing technologies in functional genomics

生科102甲 498062076 吳亞璇

advances in dna sequencing technologies
Advances in DNA sequencing technologies
  • Sanger sequencing
  • 454 sequencing technology
  • Illumina
  • ABI/SOLiD
sanger sequencing
Sanger sequencing

(1)利用DNA當模板,分別將DNA分成4組同時加入DNA polymerase以及dNTP合成。

(2)分加入有標螢光的ddATP、ddGTP、ddTTP、ddNTP反應,使合成過程中隨機終止聚合反應,造成不同大小的DNA片段。

(3)透過電泳分析讀出待測物DNA序列。

454 sequencing technology
454 sequencing technology

(1)將待測基因組序列打斷成較短的DNA片段,並使雙鏈熔解拆分為單鏈DNA分子。

(2) 利用直徑為28µm的DNA吸附珠與單鏈DNA分子吸附,然後利用乳化PCR(emulsion-based PCR , emPCR)擴增每個珠子上的DNA分子拷貝數。

(3)將每個珠子置於隔離反應井(直徑44µm)中。

(4)使用焦磷酸法測序,分析配對出完整之核酸序列。

illumina
Illumina

(1)將待測DNA 打斷成200-500 bp的小片段,並於兩端接上轉接序列。

(2)將已接上轉接序列的待定序基因小片段,置入表面帶有互補轉接序列的晶片,透過橋式聚合酶鏈鎖反應進行增幅,接著置入依不同鹼基而標記且可移除螢光分子的反應試劑。

(3)反覆進行螢光標記移除、試劑置換與偵測,以快速讀取大量之定序結果,最後輔以資訊軟體系統,即可分析配對出完整之DNA 序列。

abi solid
ABI/SOLiD

(1)將待測基因組序列打斷成較短的DNA片段,並使雙鏈熔解為單鏈。利用吸附珠與DNA分子吸附並固定DNA(控制條件使每一個珠子僅吸附一條DNA單鏈分子)

(2)利用乳化PCR(emulsion-based PCR , emPCR)擴增珠子上的DNA分子數目。

(3)採用一系列用螢光標記的8個核苷酸探針(探針3‘端的前2個核苷酸為測序反應提供信息)不同的寡核苷酸探針與模板序列競爭結合,只有3’端前2個核苷酸與模板鏈相應位置完全配對互補,探針才會被連接到模板的互補鏈上。

(4)通過多輪反應,模板鏈上位置編號為(1 or 2 +8N)的核苷酸位點的序列將被測定。如此反覆幾次,就可以獲得模板DNA分子的全長序列信息。

making use of next generation sequencer data format pains and gains of plentiful short reads
Making use of next-generation sequencer data format: pains and gains of plentiful short reads
  • 利用short reads(tags)來分析DNA分子的起源
  • 似基因表現連續法(Serial analysis of gene expression,SAGE)
  • 常用於分析基因轉錄部分(如gene expression, RNA profiling)
  • 近來也用於偵測蛋白質在RNA染色質上的binding site
  • 用於miRNAs和siRNAs還未成熟
transcriptome sequencing by next generation technologies
Transcriptome sequencing by next-generation technologies
  • 分析cDNA而不是DNA,因此可減少序列的大小
  • 用於基因 geneexpression profiling、gene annotation和rearrangement detection to noncoding RNA discovery and qualification
  • 可做多方面的用途,如了解the level of gene expression、the structure of genomic loci和

sequence variation present at loci (Ex: SNPs)

  • 目前多用454 technology,但近來也有用Illumina來做transcriptomics。
slide13
Application of next generation sequencing for analysis of epigenetic modification of histones and DNA
  • 利用next-generation來分析DNA和染色質的共價連接
  • 用於了解oncogenesis的發展
  • 也用於human epigenome project (HEP)
  • next-generationsequencing 加速epigenomic research的進行
concluding remarks
Concluding remarks
  • Next-generation sequencing 目前已成為主要的genomic technology
  • 可提供genome-scale sequencing capacity而非一定要genome centers
  • 由於讀取的序列較短,mapping efficiency也會較高
  • 由於cloning-freeamplification技術的發達,目前單股的DNA即可定序
  • 儘管目前定序技術日趨發達,但方法的發展仍屬嬰兒期,善須經由許多研究來奠定這些方法的完備性。