NGS Library Preparation Method for Transcriptome Profiling With Enhanced Sensitivity of Transcript Detection

Transcriptome profiling by Next Generation Sequencing (RNA-seq) is a powerful technique for large-scale genome-wide expression analysis, including mRNA, non-coding RNA, and miRNA profiling. RNA-seq is beginning to have multiple applications in clinical diagnostics; providing better insights into how altered RNA expression impacts the biological pathways and the molecular mechanisms associated with disease progression. The successful adoption of NGS into the clinical laboratory will depend on the library preparation techniques that can capture the entire molecular repertoire within a sample without any sequence bias. Here, we present a method for directional or strand-specific RNA-seq that retains information about which strand of DNA is expressed. Determining the polarity of RNA transcripts is important for the correct annotation of novel genes, identification of antisense transcripts with potential regulatory roles, and for correct determination of gene expression levels in the presence of antisense transcripts.  This method is based on the labeling and excision of the second strand cDNA, and it is compatible with both poly A-tail enriched and ribosome-depleted RNA. We will provide a comprehensive analysis of different eukaryotic and prokaryotic RNA libraries including library performance, library complexity, duplication rate, and continuity of gene coverage and strand specificity. Our results show this improved method generates high library yields that allow use of low amount of input RNA with reduced PCR duplicates, delivering high quality strand-specific RNA-seq data. This streamlined protocol is also amenable to large-scale library construction and automation.