Project description:In this study, we present a comprehensive evaluation of four RNA-Seq library preparation methods. We used three standard input protocols, the Illumina TruSeq Stranded Total RNA and TruSeq Stranded mRNA kits, and a modified NuGEN Ovation v2 kit; and an ultra-low-input RNA protocol, the TaKaRa SMARTer Ultra Low RNA Kit v3. Our evaluation of these kits included quality control measures such as overall reproducibility, 5’ and 3’ end-bias, and the identification of DEGs, lncRNAs, and alternatively spliced transcripts. Overall, we found that the two Illumina kits were most similar in terms of recovering DEGs, and the Illumina, modified NuGEN, and TaKaRa kits allowed identification of a similar set of DEGs. However, we also discovered that the Illumina, NuGEN and TaKaRa kits each enriched for different sets of genes.
Project description:In this study, we present a comprehensive evaluation of four RNA-Seq library preparation methods. We used three standard input protocols, the Illumina TruSeq Stranded Total RNA and TruSeq Stranded mRNA kits, and a modified NuGEN Ovation v2 kit; and an ultra-low-input RNA protocol, the TaKaRa SMARTer Ultra Low RNA Kit v3. Our evaluation of these kits included quality control measures such as overall reproducibility, 5’ and 3’ end-bias, and the identification of DEGs, lncRNAs, and alternatively spliced transcripts. Overall, we found that the two Illumina kits were most similar in terms of recovering DEGs, and the Illumina, modified NuGEN, and TaKaRa kits allowed identification of a similar set of DEGs. However, we also discovered that the Illumina, NuGEN and TaKaRa kits each enriched for different sets of genes.
Project description: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.
Project description:We evaluated the performance of 5 library prep protocols by using total mRNA and IP RNA of mouse liver,we found all the 5 library preparation kits detect more enrichment effects than depletion effect. The profiles being generated by SMARTer kit is different than all other kits.