基因转录组的测定及分析培训课件.ppt

中科院北京基因组研究所 * 后续工作和展望 进一步分析这些在内含子以及基因间区域的表达，并且用实验证明它们的真实表达。 在生化和细胞水平进一步研究rad9的功能，主要的一个方向是rad9对细胞表观遗传学的调控。利用chip-seq方法研究rad9基因敲除后染色质的状态变化，以及研究rad9基因敲除后DNA水平甲基化状态的变化。 进一步挖掘细胞在毒力的作用下基因表达谱的变化，构建相关基因的调控网络，并研究细胞在毒力的作用下表观遗传学水平上的变化。 Transcriptome de novo assembly RNA-Seq Denovo Mapping (Bowtile, MAQ) with reference Assembly (SOAPdeno, velvet, Oases) no reference Combine Assembly and Mapping with references but reference sequences is not fine Hot topic – fuse gene Sample collection GS FLX sequencing Rapid library cDNA synthesis RNA fragment mRNA isolation RNA extraction Assembly Gene annotation Raw reads Isotigs Pathway analysis GO analysis Liquid nitrogen DNase I treated Rapid library adaptor Random primer rRNA depletion Gene difference expression analysis Newbler 2.5 BlastX BlastN InterPro Data assessment emPCR enrichment 454 RNA-seq processes 454 isotig graphic 454 reads runassembly -v –s (remove primer, vector, low quallity region(q13), high coverage(20x)) Contig + Singleton Contig + Singleton Contig + Singleton Contig + Singleton Contig + Singleton Seqclean (short reads remove(100nt)) Contamination remove(microbial i90% l90%) Allele transcripts remove(i99% l-dif 10nt) Alternative splicing remove() Contig + Singleton Homopolymer remove(solexa,solid) Contig + Singleton Scaffold construct ion(cDNA meta-paired) Scaffold construct ion(cDNA meta-paired) Contig + Singleton Contig + Singleton Sequence Extension Unigene 1. Identification of genes (De novo transcriptome only)2. Structure of transcripts: Identification of untranslated region (UTR), boundary of intron,alternative splicing and start codon, etc.3. Identification of non-coding unit: Non-coding RNA, precursor of microRNA, etc.4. Determing gene expression in transcriptional level5. Identification of new transcription unit Application of RNA-Seq Capacity RNA-Seq Detected signals Digital signals Detected range Nearly all the transcripts Detected accuracy From several copies to 100,000 copies Resolutio