title: Comparison of CAGE and RNA-seq transcriptome profiling using clonally amplified and single-molecule next-generation sequencing.
abstract: CAGE (cap analysis gene expression) and RNA-seq are two major technologies used to identify transcript abundances as well as structures. They measure expression by sequencing from either the 5' end of capped molecules (CAGE) or tags randomly distributed along the length of a transcript (RNA-seq). Library protocols for clonally amplified (Illumina, SOLiD, 454 Life Sciences [Roche], Ion Torrent), second-generation sequencing platforms typically employ PCR preamplification prior to clonal amplification, while third-generation, single-molecule sequencers can sequence unamplified libraries. Although these transcriptome profiling platforms have been demonstrated to be individually reproducible, no systematic comparison has been carried out between them. Here we compare CAGE, using both second- and third-generation sequencers, and RNA-seq, using a second-generation sequencer based on a panel of RNA mixtures from two human cell lines to examine power in the discrimination of biological states, detection of differentially expressed genes, linearity of measurements, and quantification reproducibility. We found that the quantified levels of gene expression are largely comparable across platforms and conclude that CAGE and RNA-seq are complementary technologies that can be used to improve incomplete gene models. We also found systematic bias in the second- and third-generation platforms, which is likely due to steps such as linker ligation, cleavage by restriction enzymes, and PCR amplification. This study provides a perspective on the performance of these platforms, which will be a baseline in the design of further experiments to tackle complex transcriptomes uncovered in a wide range of cell types.
authors.affiliation: RIKEN Preventive Medicine and Diagnosis Innovation Program, Saitama 351-0198, Japan;
authors.forename: Hideya
authors.initials: H
authors.lastname: Kawaji
authors.forename: Marina
authors.initials: M
authors.lastname: Lizio
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Itoh
authors.forename: Mutsumi
authors.initials: M
authors.lastname: Kanamori-Katayama
authors.forename: Ai
authors.initials: A
authors.lastname: Kaiho
authors.forename: Hiromi
authors.initials: H
authors.lastname: Nishiyori-Sueki
authors.forename: Jay W
authors.initials: JW
authors.lastname: Shin
authors.forename: Miki
authors.initials: M
authors.lastname: Kojima-Ishiyama
authors.forename: Mitsuoki
authors.initials: M
authors.lastname: Kawano
authors.forename: Mitsuyoshi
authors.initials: M
authors.lastname: Murata
authors.forename: Noriko
authors.initials: N
authors.lastname: Ninomiya-Fukuda
authors.forename: Sachi
authors.initials: S
authors.lastname: Ishikawa-Kato
authors.forename: Sayaka
authors.initials: S
authors.lastname: Nagao-Sato
authors.forename: Shohei
authors.initials: S
authors.lastname: Noma
authors.forename: Yoshihide
authors.initials: Y
authors.lastname: Hayashizaki
authors.forename: Alistair R R
authors.initials: AR
authors.lastname: Forrest
authors.forename: Piero
authors.initials: P
authors.lastname: Carninci
mesh_terms: D020869:Gene Expression Profiling; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D012313:RNA / Q000235:genetics*; D017423:Sequence Analysis, RNA / Q000379:methods; D059467:Transcriptome / Q000235:genetics*
chemical_list: D012313:RNA