下一代测序原理

Cleavage
Fluorescent dye labeled nucleotides are cleaved from hybridized probe.
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UCSC Sequencing Center
2nd Ligation Cycle
The next set of labeled probes hybridize and are ligated to the previously hybridized probe.
Every 5th base is interrogated
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UCSC Sequencing Center
Reset
Template is stripped
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UCSC Sequencing Center
1st cycle after reset
图7 差异表达基因等级聚类图
表达模式相似的基因通常具有相似的功能。我们利用cluster软件（Eisen, Spellman, et al. 1998），以欧 氏距离为距离距阵计算公式，对差异表达基因和实验条件同时进行等级聚类 分析，聚类结果用javaTreeview显示（Saldanha. 2004）。图7中每列代表一个实验条件，每行代表一个基 因，不同表达变化倍数用不同颜色表示，红色表示表达上调，绿色表示表达下调。
De-phosphorylation
A phosphatase cleaves the 3’-phosphate from the universal sequencing primer preventing ligation to templates where no probe was ligated.
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UCSC Sequencing Center
2nd Cycle Visualization
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UCSC Sequencing Center
2nd cycle cleavage
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UCSC Sequencing Center
DGE 数字表达谱
图2 Tag制备的原理和步骤。利用Oligo (dT)的beads富集总RNA中mRNA，并逆转录为双 链cDNA，采用4碱基识别酶NlaIII酶切双链cDNA，链接Illumina adapter1，利用MmeI酶 切3'端CATG下游17bp碱基，并在3'端链接Illumina adapter2。再加入Primer GX1和 Primer GX2进行PCR扩增。扩增后样本通过6% TBE PAGE胶回收85碱基条带，纯化后通 过Illumina基因表达测序法测序。
第二代测序技术原理及数据分析
SOLiD Sequencing Chemistry
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UCSC Sequencing Center
4-color Ligation Reaction
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UCSC Sequencing Center
A complementary dye-labeled probe hybridizes is ligated to the universal sequencing primer.
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UCSC Sequencing Center
qRT-PCR验证结果
RNA-Seq结果与qRT-PCR具有高度一致性
随机选取15个基因进行qRT-PCR验证
发现12个基因qRT-PCR结果与RNA-Seq相一致
UCSC Sequencing Center
2nd round
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UCSC Sequencing Center
Contig
• Contig (from contiguous) is a set of overlapping DNA segments derived from a single genetic source.
Genome
73
Scafolds
Contigs
差异基因编码的蛋白质之间相互作用
相互作用网络分析整合了BIND、BioGrid、HPRD等相互作用网络数据库的信息，结果 文件中的网络由差异表达基因以及跟差异表达基因有直接相互作用的基因组成。结果 文件可用Medusa软件（Hooper, Bork. 2005）显示。
A new set of universal sequencing primers is hybridized, offset by (n-1).
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UCSC Sequencing Center
1st cycle after reset
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Fra Baidu bibliotek
基因表达量分布
浆果发育三个阶段共有、特有基因表达分布图
发现385个基因具有AS（alternative splicing），其中 210个基因只在一个浆果发育stage发生AS，有97个基因 在2个stage发生AS，而有78个基因在研究的浆果的3个发 育stages都发生了AS。 鉴定了新的基因，发现了glutathione-S-transferase (GST)家族的53个新基因，这些基因是芯片没有检测到 的。MYB转录因子家族中28个新基因，这些基因因为 表达丰度低、缺少探针序列而无法被芯片检测到。
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UCSC Sequencing Center
Visualization
The fluorescently labeled dyes attached to each probe are visualized.
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UCSC Sequencing Center
Overlapping Reads
ACGTACTACTTCACCGAAAC GTACTACTTCACCGAAACGA TACTTCACCGAAACGATCGTT TTCACCGAAACGATCGTTAC CGAAACGATCGTTACGATCC ACGATCGTTACGATCCATCC TTACGATCCATCCCTACCGC ||||||||||||||||||||||||||||||||||||||||||||| ACGTACTACTTCACCGAAACGATCGTTACGATCCATCCCTACCGC Contig