GSE124167
Comparative evaluation of RNA-Seq library preparation methods for strand-specificity and low input
Library preparation is a key step in gene expression quantification. There are considerable advantages to both strand specific sequencing and the ability to sequence samples with very small amounts of starting material. Until recently there was no kit available that allowed both simultaneously. The standard Illumina-TruSeq stranded mRNA Sample Preparation kit requires abundant starting quantity while the Takara Bio-SMART-Seq® v4 Ultra® Low Input RNA kit allows for ultra low starting quantities but sacrifices strand specificity. Recently a kit that can do both, SMARTer® Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian by Takara Bio, has become available. Evaluating the performance and effects of these sample preparation kits is a critical determinant for selecting the appropriate sequencing protocol, but a comprehensive comparison is currently missing. To address this we performed a detailed comparative analysis of sequencing libraries prepared with the three kits. We prepared a set of samples representing two experimental conditions with each kit, allowing for comparison of the kits in a standard realistic differential expression analysis. We find substantial differences in the levels of alignment and differential gene expression. Using differential expression analysis we show that using Pico results in identifying 55% less differentially expressed genes than TruSeq. Nevertheless, using gene pathway enrichment analysis we find similar results for all three kits, indicating that ultimately comparable functional results can be reached. Overall design: Liver RNA from mice treated with IL-1ß or saline. Both treatment conditions have 3 biological replicates.