Automated workflow for RNA-Seq analysis: application and testing with various types of RNA-Seq protocols

Abstract

► Protocols for creating libraries for RNA-Seq using Illumina’s nextgeneration sequencing platforms include preps that utilize either poly-A selected mRNA or Total RNA. ► In addition the preps can also be performed with different types of cDNA synthesis chemistries, some of which are non-stranded and some that preserve the strandedness of the mRNA during cDNA creation, like RNA ligation methods or the incorporation of modified bases like dUTP. ► There are total RNA prep methods that use the RiboZeroTM kit (Epicentre) or the Duplex Specific Nuclease (DSN, from Evrogen) for rRNA reduction. Both of these approaches allow one to make full-length cDNA libraries that are completely independent of the integrity and degradation of the starting RNA material. Since these Total RNA-Seq methods do not use a poly-A selection procedure they allow us to sequence all of the RNA in the library including many non-coding RNAs that can be missed in other gene expression assays. Automated Workflow for RNA-Seq Data Analysis