• Document: Differential gene expression analysis using RNA-seq
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Differential gene expression analysis using RNA-seq Applied Bioinformatics Core, August 2017 Friederike Dündar with Luce Skrabanek & Ceyda Durmaz https://abc.med.cornell.edu/ Day 1: Introduction into high-throughput sequencing [many general concepts!] 1.  RNA isolation & library preparation 2.  Illumina’s sequencing by synthesis 3.  raw sequencing reads • download • quality control 4.  experimental design RNA-seq is popular, but still developing “RNA%seq)is)not$a$mature$technology.)It$is$ undergoing$rapid$evolution$of)biochemistry) of)sample)preparation;)of)sequencing) platforms;)of)computational)pipelines;)and)of$ subsequent$analysis$methods$that$include$ statistical$treatments)and)transcript)model) building.)“) ENCODE&consortium& Reuter et al. ( 2015). Mol Cell. Goodwin, McPherson & McCombie (2016). Nat Gen, 17(6), 333–351 “Analysis paralysis” •  basically no generally accepted standard reference •  myriad tools ! highly complex & specialized “pipelines” “The (…) flexibility and seemingly infinite set of options (…) have hindered its path to the clinic. (…) The fixed nature of probe sets with microarrays or qRT-PCR offer an accelerated path (…) without the lure of the latest and newest analysis methods.” Byron et al., 2016 Byron et al. Nat Rev Genetics (2016) What to expect from the class Sample type Experimental design •  Controls & quality •  No. of replicates •  Randomization NOT COVERED: •  novel transcript Library preparation discovery Biological question •  Poly-A enrichment •  transcriptome vs. ribo minus •  Expression quantification assembly •  Strand information •  Alternative splicing •  De novo assembly needed •  alternative splicing •  mRNAs, small RNAs analysis •  …. (see the course notes for Bioinformatics references to useful •  Aligner reviews) •  Normalization Sequencing •  DE analysis strategy •  Read length •  PE vs. SR •  Sequencing errors RNA-seq workflow overview Total RNA extraction cells RNA •  Fragmentation fragments •  mRNA enrichment •  Library preparation cDNA with adapters Sequencing •  Cluster generation •  Sequencing by synthesis •  Image acquisition Bioinformatics Quality control of RNA extraction 28S:18S ratio avoid degraded RNA junk RNA-seq library preparation 16 EX PE R IM EN TA L C ELL RE S EA RC H 3 22 (2014) 12 –20 RNA extraction

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