Using Synthetic Mouse Spike-In Transcripts to Evaluate RNA-Seq Analysis Tools

PLoS One. 2016 Apr 21;11(4):e0153782. doi: 10.1371/journal.pone.0153782. eCollection 2016.


One of the key applications of next-generation sequencing (NGS) technologies is RNA-Seq for transcriptome genome-wide analysis. Although multiple studies have evaluated and benchmarked RNA-Seq tools dedicated to gene level analysis, few studies have assessed their effectiveness on the transcript-isoform level. Alternative splicing is a naturally occurring phenomenon in eukaryotes, significantly increasing the biodiversity of proteins that can be encoded by the genome. The aim of this study was to assess and compare the ability of the bioinformatics approaches and tools to assemble, quantify and detect differentially expressed transcripts using RNA-Seq data, in a controlled experiment. To this end, in vitro synthesized mouse spike-in control transcripts were added to the total RNA of differentiating mouse embryonic bodies, and their expression patterns were measured. This novel approach was used to assess the accuracy of the tools, as established by comparing the observed results versus the results expected of the mouse controlled spiked-in transcripts. We found that detection of differential expression at the gene level is adequate, yet on the transcript-isoform level, all tools tested lacked accuracy and precision.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Computational Biology
  • Gene Expression Profiling / standards*
  • High-Throughput Nucleotide Sequencing / standards*
  • Mice
  • Quality Control
  • RNA, Messenger / genetics*
  • Reference Standards
  • Sequence Analysis, RNA / standards*


  • RNA, Messenger

Grants and funding

This project was supported by a grant from Weizmann–Staff Scientists Internal Grant Program (D.L., E.F. and S.H.S.) []. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.