Abstract 3185: Rapid and efficient methods for preparing rRNA-depleted and directional RNA-Seq libraries from low-input and FFPE RNA samples

Abstract

Abstract Massively parallel sequencing of cDNA libraries (RNA-Seq) is rapidly becoming the preferred method for transcript profiling, and analysis of novel transcripts, novel isoforms, alternative splice sites, rare transcripts and cSNPs, compared to microarrays. Preparing NGS RNA-Seq libraries typically require first isolating rRNA-depleted RNA or enriching for poly(A)+ mRNA. However, total RNA isolated from formalin-fixed, paraffin-embedded (FFPE) patient tissue samples is normally fragmented, making it not suitable for rRNA-depletion using some commercially available kits or poly(A)+ enrichment, which results in a 3′ sequence bias. Here, we present an efficient “single-pass” rRNA-depletion method (Ribo-Zero™ technology) for use with as little as 100 ng total RNA input from either intact or fragmented (e.g., FFPE) RNA samples. Additionally, an improved, more user-friendly version of the ScriptSeq™ RNA-Seq method (ScriptSeq™ v2) is used to rapidly prepare directional (∼99% strandedness) RNA-Seq libraries in about 2.5 hours, in a single-tube workflow, from either the intact or fragmented Ribo-Zero™ treated RNA samples. The ScriptSeq™ method does not require end-polishing, adaptor-ligation, cDNA fragmentation, or gel-size selection. The combined Ribo-Zero™ and ScriptSeq™ workflow is completed in about 5 hours, generating cluster-ready NGS libraries that contain <2 % of reads that map to rRNA sequences (nuclear- and mitochondrial-encoded) and ∼98% of reads that map to the genome, while maintaining the representation of coding and non-coding transcripts, independent of polyadenylation. This reduction in rRNA sequence reads improves sequence depth and coverage of mRNA, and increases the percentage of uniquely mapped reads required for transcriptome analysis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3185. doi:1538-7445.AM2012-3185