摘要:mRNA-seq是通过逆转录过程将细胞产生的RNA转化为DNA (cDNA,互补,并对获得的cDNA进行文库构建)。然后对得到的DNA进行测序,并从观察到的特定DNA丰度中,从中推断细胞中mRNA的原始量。大多数mRNA-seq分析的目标是找到在实验条件下转录水平发生变化的基因或转录本,即差异表达。通过找到这些基因和转录本,我们可以推断出不同条件的功能特征。mRNA-seq通常建议的每种状态最小生物学重复次数为3次,5次更好。如果预期到结果可能差异微妙或者生物变异显着时 (例如对活体动物进行实验),需要更多的重复。所以,一组实验 (对照组和实验组) 意味着会有6~10个样品,产生6~10个或者更多fastq文件。所以能够明确以及流程化分析过程十分重要。
关键词: mRNA-seq analysis, 序列比对, 测序定量, 差异表达
仪器设备
软件
实验步骤
注:本教程是基于已经在服务器上和个人电脑上安装好相关软件进行的。如果安装出现问题,请参考以下链接: FastQC:https://www.bioinformatics.babraham.ac.uk/projects/download.html mulitiQC:https://multiqc.info/ flexbar:https://github.com/seqan/flexbar STAR:https://github.com/alexdobin/STAR Samtools:http://www.htslib.org/download/ HTSeq:https://htseq.readthedocs.io/en/release_0.10.0/
参考文献
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