Dataset Information

Systematic evaluation of RNA-Seq preparation protocol performance (RNASeq: SMARTer)

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE131396 | GEO | 2019/06/27

REPOSITORIES: GEO

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Project description:Purpose: The goals of this study is to determine the best method of gene expression quantification (RNA-seq, Microarray, NanoString) and amplification kits adapted to low-input and/or low-quality RNA samples (FFPE samples) Methods: Mouse bladder cancer cell line (mouse bladder cancer cell line, BC57) and mouse normal mouse normal urothelium were fixed in formalin and embedded in paraffin (FFPE), andfesh frozen (FF) in liquid nitrogen. The total RNA of these 4 samples were tested in triplicate by 3 technologies (NanoString, RNA-seq and Microarray) and the results were compared to its reference (high-quality and high-input RNA of mouse bladder cancer cell line and mouse normal mouse normal urothelium). For RNA-sequencing, each sample was tested by two library contruction kits: SMARTer Stranded Total RNA-Seq Kit - Pico Input mammelian and Ovation SoLo NuGEN RNA-seq System with three input quantities: 50pg, 250pg and 2ng of total RNA, except for the SMARTer Stranded Total RNA-Seq Kit - Pico Input mammelian kit for which the minimum recommended quantity was 250pg of total RNA. RNA-seq based on poly(A) tail enrichment was considered as the reference and was done for the two FF samples at high amount (1µg of total RNA) . Results obtained with the two tested kits were compared to those based on poly(A) tail enrichment. To determine which is the best kit suitable for RNA-seq, low-input and low-quality RNA samples, we performed RNA-seq control quality metrics, principal component analysis, and a differential analysis between the mouse bladder cancer cell lines and the mouse normal mouse normal urothelium for each input quantity, library kit and method of sample preservation (FF or FFPE). Results: The Ovation SoLo NuGEN RNA-seq System library kit are recommended for quantification of gene expression of FFPE and FF samples at low-input (down to 50pg) whereas the SMARTer Stranded Total RNA-Seq Kit - Pico Input mammelian is only suitable for FF samples from 250pg of total RNA.

Dataset Information

Systematic evaluation of RNA-Seq preparation protocol performance (RNASeq: SMARTer)

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets