Comprehensive Capture of FFPE RNA for RNA-Seq Using a Continuous Synthesis Chemistry and Post-Library Ribodepletion

The most common archived biospecimen in oncology, formalin-fixed paraffin-embedded (FFPE) tissue is considered a key source material for gene expression profiling of tumors using RNA sequencing (RNA-Seq). However, FFPE RNA is highly degraded and is therefore suboptimal for most currently available RNA-Seq library preparation workflows, which also focus on capturing only long (>200 bp) RNAs. In this study, we present a novel RNA-Seq, library preparation workflow (Figure 1) that overcomes many of the challenges associated with generating complex, rich data from FFPE RNA and other sample types with scarce and/or degraded RNA. This solution, the SEQuoia Complete Stranded RNA Library Prep Kit, leverages a proprietary retrotransposon enzyme in place of the traditional reverse transcriptase and ligases found in other RNA-Seq library preparation kits, enabling a continuous synthesis reaction that converts RNA to complementary DNA (cDNA) and adds both sequencing adapters in a single step. Our data show that using this kit in conjunction with the SEQuoia RiboDepletion Kit (for post-library ribodepletion) yields a more complex library from FFPE RNA that includes both long (>200 bp) and short (<200 bp) RNA biotypes and is therefore more representative of the complete transcriptome.

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