转录组测序原理.pptVIP

鉴定融合基因 新转录本预测 Genomic intergenic region Reads cluster Paired Reads distribution Paired-End (PE) Reads N Eng J Med 2009 SNP分析 Deep RNA sequencing at single base-pair resolution reveals high complexity of the rice transcriptome Genome Res 2010 Rice Transcriptome Material callus root at seedling stage（14d） shoot at seedling stage（14d） flag leaves（2 stages） panicle（3 stages） Methods RNASeq（paired-end single end） DGE small RNA（18-30 nt） 基因功能注释 基因结构分析 鉴定出大量新转录本 可变剪接鉴定 基因融合鉴定 无参考基因组生物信息分析 Unigene功能注释 Unigene的GO分类 Unigene代谢通路分析 预测编码蛋白框（CDS） Unigene表达差异分析 Unigene在样品间的差异GO分类和Pathway富集性分析 De novo reads组装流程 Unigene GO 分类 Unigene COG 功能分类 基因表达差异分析 N1:total tag Number in sample A N2:total tag Number in sample B X :Gene expression level in sample A y :Gene expression level in sample B Reference: Audic S. et al. The significance of digital gene expression profiles. Genome Res. 1997 7(10):986-995 Unigene pathway 富集性分析 Pathway富集性分析列表 Thank you 指一个细胞内基因组DNA转录得到的所有转录产物以及转录物在细胞特定发育时期或特定生理条件下的表达水平 转录本主要包括mRNA，small RNA，non-coding RNA * 反转录时容易丢失mRNA5′端的信息，mRNA片段越长所受影响越大，打断成200nt的片段，可减少5′端的信息丢失。 mRNA的二级结构也会屏蔽随机引物的结合位点，通过打断可减少二级结构的数量。 * Fragmentation of oligo-dT primed cDNA is more biased towards the 3′ end of the transcript. RNA fragmentation provides more even coverage along the gene body, but is relatively depleted for both the 5′ and 3′ ends * * SBS:边合成边测序 于2009年发表在N Eng J Med， 利用转录组测序技术研究了4例卵巢颗粒细胞瘤(GCT)和11例其它卵巢癌患者病变组织，发现FOXL2基因 402C→G错义突变仅在4例GCT发生，不在49例其他类型的性索间质肿瘤中发生，不在329例和卵巢无关的肿瘤和乳腺癌中发生 Eight organs from different developmental stages were collected from this strain of rice, including callus, root at seedling stage of 14 d, shoot at seedling stage of 14 d, flag leaves at tillering stage, flag leaves at flowering stage, panicle at booting stage, panicle at flowering stage, and anicle at filling stage. we present the first transcriptome atlas for eight organs of cultivated rice. Using high-throughput pai