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RNA-Seq分析:背景及前沿技术介绍
Junyi Li, PhD
jyli@
Key Lab of Systems Biology
Shanghai Institute for Biological Sciences
Typical RNA-Seq experiment
Nat Rev Genet. 10:57
Why sequence RNA
• Functional genomics studies
– Genome may be constant but an experimental condition has a
pronounced effect on gene expression
– Many gene features can only be observed at the RNA level:
alternative isoforms, fusion transcripts, RNA editing
– Interpreting regulatory mutations that do not have an obvious effect
on protein sequence: promoters, splice sites
Advantage of RNA-Seq
Nat Rev Genet. 10:57
Challenge in RNA-Seq
• Sample
– Purity?, quantity?, quality?
– RNA is more easily degraded compared to DNA
• RNAs come in a wide range of sizes
– Small RNAs must be captured separately
– PolyA selection of large RNAs may result in 3’
• The relative abundance of RNAs vary wildly
– 105 – 107 orders of magnitude
– Since RNA sequencing works by random sampling, a small fraction of highly expressed
genes may consume the majority of reads
– Ribosomal and mitochondrial genes
Experiment design consideration
• Standards, Guidelines and Best Practices for RNA-seq
– The ENCODE Consortium
– Download from the Course Wiki
– Meta data to supply, replicates, sequencing depth, control
experiments, reporting standards, etc.
• /courses/2013/cbw/ENCODE_RNAseq_standards_v1.0.pdf
RNA quality
• The RNA integrity number (RIN) is an algorithm for assigning integrity values
to RNA measurements.
• It is applied to electrophoretic RNA measurements and based on a combination
of different featur
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