Abstract
RNA-seq is a powerful tool used to obtain in-depth information on expression profiling, gene annotation, and transcript discovery. With the growing popularity of RNA sequencing, new library preparation techniques are becoming commercially available. These techniques are improvements on the classic poly-A selection and rRNA reduction methods, and in some cases sensitive enough to analyze the transcriptome of a single cell. However, limited information is available on comparative analysis of these methods and their appropriate application for the transcriptome studies. We utilized Illumina's HiSeq technology to compare the merits of four commercial sample preparation kits: NuGen's Ovation RNA-seq system v2, Illumina's TruSeq RNA Sample Preparation kit v2, Epicentre's ScriptSeq RNA-seq kit v2 and Clontech's SMARTer Ultra Low RNA kit. We found that the quality of input RNA was critical for optimum performance of SMARTer Ultra Low RNA kit. Ovation and ScriptSeq kits, on the other hand, worked well with moderate quality input RNA as well. Based on analysis of the sequencing data, 12% of reads from ScriptSeq mapped to the mitochondrial genes as compared to 24% reads from Ovation. The library complexity and percentage of reads aligning to non-exonic region was similar between both kits. However, 28% reads aligned to the coding region for ScriptSeq versus 18% for Ovation. While TruSeq and SMARTer kits are designed for Poly-A containing RNAs only, ScriptSeq and Ovation kits provide more global analysis of the transcriptome. Analyzing the differences between these methods provides a better understanding of their specific advantage over the other. This information is especially useful for Sequencing Core Facilities, to recommend and apply appropriate methods to different transcriptome studies.